Fibronectin based scaffold domain proteins that bind PCSK9

ABSTRACT

The present invention relates to fibronectin based scaffold domain proteins that bind PCSK9. The invention also relates to the use of the innovative proteins in therapeutic applications to treat atherosclerosis, hypercholesterolemia and other cholesterol related diseases. The invention further relates to cells comprising such proteins, polynucleotides encoding such proteins or fragments thereof, and to vectors comprising the polynucleotides encoding the innovative protein.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted 5 in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 13, 2011, isnamed 20110413_SEQT_11594USNPST25.txt and is 1,382 KB in size.

FIELD OF THE INVENTION

The present invention relates to fibronectin based scaffold domainproteins that bind proprotein convertase subtilisin kexin type 9(PCSK9). Further, the use of the innovative proteins in therapeuticapplications to treat atherosclerosis, hypercholesterolemia and othercholesterol related diseases, cells comprising such proteins,polynucleotides encoding such proteins or fragments thereof, and vectorscomprising the polynucleotides encoding the innovative proteins aredescribed herein.

INTRODUCTION

Atherosclerosis is a disease of the arteries responsible for coronaryheart disease (CHD) that underlies most deaths in industrializedcountries (Lusis (2000)). Several risk factors for CHD have now beenwell established: dyslipidemias, hypertension, diabetes, smoking, poordiet, inactivity and stress. The most clinically relevant and commondyslipidemias are characterized by an increase in low densitylipoprotein and very low density lipoproteins (LDL and VLDL) withhypercholesterolemia in the absence or presence of hypertriglyceridemia(Fredrickson et al. (1967)). An isolated elevation of LDL cholesterol isone of the most common risk factors for CHD. PCSK9 (also referred to asHCHOLA3, NARC-1, or FH3) is a protease belonging to the proteinase Ksubfamily of the secretory subtilase family (Naureckiene et al., Arch.Biochem. Biophy., 420:55-57 (2003)). PCSK9 has been shown to be a keyregulator of cholesterol homeostasis and circulating low densitylipoprotein levels. Circulating PCSK9 protein controls LDL metabolism bydirectly binding to the LDL receptor and promoting its degradation inhepatocytes. PCSK9-mediated down-regulation of LDL receptor protein andactivity leads to reduced clearance of LDL from the circulation andhigher LDL levels. Several mutant forms of PCSK9 are known, includingS127R, N157K, F216L, R218S, and D374Y, with S127R, F216L, and D374Ybeing linked to autosomal dominant hypercholesterolemia (ADH). It isbelieved that wild-type PCSK9 increases the turnover rate of the LDLreceptor causing lower LDL clearance (Maxwell et al., Proc. Natl. Acad.Sci., 102(6):2069-2074 (2005); Benjannet et al. and Lalanne et al.),while PCSK9 loss of function mutations result in increased levels of lowdensity lipoprotein receptor (LDLR), increased clearance of circulatingLDL, and a corresponding decrease in plasma cholesterol levels (Rashidet al., Proc. Natl. Acad. Sci., 102(15):5374-5379 (2005)). As such,PCSK9 is a potential target for the treatment of atherosclerosis,hypercholesterolemia and other cholesterol related diseases. Particularepitopes of PCSK9, PCSK9 binding molecules and uses thereof aredescribed in WO 2008/125623.

Fibronectin based scaffolds are a family of proteins capable of evolvingto bind any compound of interest. These proteins, which generally makeuse of a scaffold derived from a fibronectin type III (Fn3) or Fn3-likedomain, function in a manner characteristic of natural or engineeredantibodies (that is, polyclonal, monoclonal, or single-chain antibodies)and, in addition, possess structural advantages. Specifically, thestructure of these antibody mimics has been designed for optimalfolding, stability, and solubility, even under conditions that normallylead to the loss of structure and function in antibodies. An example offibronectin-based scaffold proteins is Adnectins (Adnexus, aBristol-Myers Squibb R&D Company).

Fibronectin type III (Fn3) domains comprise, in order from N-terminus toC-terminus, a beta or beta-like strand, A; a loop, AB; a beta orbeta-like strand, B; a loop, BC; a beta or beta-like strand C; a loopCD; a beta or beta-like strand D; a loop DE; a beta or beta-like strand,E; a loop, EF; a beta or beta-like strand F; a loop FG; and a beta orbeta-like strand G. Any or all of loops AB, BC, CD, DE, EF and FG mayparticipate in target binding. The BC, DE, and FG loops are bothstructurally and functionally analogous to the complementaritydetermining regions (CDRs) from immunoglobulins. U.S. Pat. No. 7,115,396describes Fn3 domain proteins wherein alterations to the BC, DE, and FGloops result in high affinity TNFα binders. U.S. Publication No.2007/0148126 describes Fn3 domain proteins wherein alterations to theBC, DE, and FG loops result in high affinity VEGFR2 binders.

It would be advantageous to obtain improved fibronectin domain scaffoldproteins that bind PCSK9 for the therapeutic treatment ofatherosclerosis, hypercholesterolemia and other cholesterol relateddiseases.

SUMMARY OF THE INVENTION

The application provides Adnectins against human PCSK9. Specifically,the invention provides for polypeptides comprising a Fn3 domain, whereinthe FG loop comprises a sequence according to the formula EX₄X₁X₅X₁X₁X₆GYX₄HR (SEQ ID NO: 451), wherein X₁ is any amino acid; X₄ is Y or F;X₅ is Y, F, or W; and X₆ is S or A. The Fn3 domain is an Fn3 domainderived from the wild-type tenth module of the human fibronectin typeIII domain (¹⁰Fn3). In some embodiments, the ¹⁰Fn3 polypeptide of theinvention is at least 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%identical to the human ¹⁰Fn3 domain.

In some embodiments, one or more loops selected from BC, DE, and FG maybe extended or shortened in length relative to the corresponding humanfibronectin loop.

The polypeptides of the invention comprise a tenth fibronectin type III(¹⁰Fn3) domain, wherein the ¹⁰Fn3 domain comprises a loop, AB; a loop,BC; a loop, CD; a loop, DE; a loop EF; and a loop FG; and has at leastone loop selected from loop BC, DE, and FG with an altered amino acidsequence relative to the sequence of the corresponding loop of the human¹⁰Fn3 domain.

In some embodiments, the polypeptide of the invention comprises an Fn3domain that comprises an amino acid sequence at least 80, 85, 90, 95,98, 99 or 100% identical to the non-loop regions.

In some embodiments, the BC loop of the protein of the inventioncomprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 2-17, 106-135, and 301-303. In certain embodiments, the BCloop of the protein of the invention comprises the italicized portion ofany one of SEQ ID NOs: 2-17, 106-135, and 301-303 as shown in Table 3.For example, in one embodiment, a BC loop comprises the sequencePPPSHGYG (residues 3-10 of SEQ ID NO: 2), DAPAHAYG (residues 3-10 of SEQID NO: 5), EPFSRLPGGGE (residues 3-13 of SEQ ID NO: 106), or DAPADGGYG(residues 3-11 of SEQ ID NO: 107).

In some embodiments, the DE loop of the protein of the inventioncomprises an amino acid sequence selected from the group consisting ofSEQ ID NOs:18-27 and 136-141. In certain embodiments, the DE loop of theprotein of the invention comprises the italicized portion of any one ofSEQ ID NOs: 18-27 and 136-141 as shown in Table 3. For example, in oneembodiment, a DE loop comprises the sequence PGKG (residues 2-5 of SEQID NO: 18), VGVG (residues 2-5 SEQ ID NO: 27), or VSKS (residues 2-5 ofSEQ ID NO: 137).

In some embodiments, the FG loop of the protein of the inventioncomprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 28-38 and 142-172. In certain embodiments, the DE loop ofthe protein of the invention comprises the italicized portion of any oneof SEQ ID NOs: 28-38 and 142-172 as shown in Table 3. For example, inone embodiment, a FG loop comprises the sequence EYPYKHSGYYHR (residues1-12 in SEQ ID NO: 28), EYPYDYSGYYHR (residues 1-12 in SEQ ID NO: 142),or EFDFVGAGYYHR (residues 1-12 in SEQ ID NO: 167).

In some embodiments, the ¹⁰Fn3 domain may begin and/or end with aminoacid substitutions, insertions or deletions.

In some embodiments, the protein of the invention comprises one loopsequence from the BC loop sequences shown in SEQ ID NOs: 2-17, 106-135,and 301-303; one DE loop sequence shown in SEQ ID NOs:18-27 and 136-141;and one FG loop sequence shown in SEQ ID NOs:28-38 and 142-172. Incertain embodiments, the protein of the invention comprises one BC loopsequence comprising the italicized portion of any one of SEQ ID NOs:2-17, 106-135, and 301-303 as shown in Table 3; one DE loop sequencecomprising the italicized portion of any one of SEQ ID NOs: 18-27 and136-141 as shown in Table 3; and one FG loop sequence comprising theitalicized portion of any one of SEQ ID NOs: 28-38 and 142-172 as shownin Table 3.

In some embodiments, the protein of the invention comprises a BC, DE andFG loop amino acid sequence at least 70, 75, 80, 85, 90, 95, 98, 99 or100% identical to of any one of SEQ ID NOS:2-38, 106-172, and 301-303.In certain embodiments, the protein of the invention comprises a BC, DEand FG loop amino acid sequence at least 70, 75, 80, 85, 90, 95, 98, 99or 100% identical to any of the italicized portions of the BC, DE, andFG loops as shown in Table 3, as described above.

In some embodiments, the anti-PCSK9 Adnectin comprises the amino acidsequence of any one of SEQ ID NOS:39-76, 173-290, and 304-309.

In some embodiments, the anti-PCSK9 Adnectin comprises the Fn3 domainamino acid sequence from position 3-96 of any one of SEQ ID NOS:39-76,173-290, and 304-309.

In one aspect, the present disclosure provides an anti-PCSK9 Adnectincomprising a BC loop having the sequence SW(X₁)_(Z)X₂G (SEQ ID NO: 323)where X₁ is any amino acid, Z is a number from 6-9, and X₂ is Y or H.

In one aspect, the present disclosure provides an anti-PCSK9 Adnectincomprising a DE loop having the sequence PX₁X₁X₁X₃T, (SEQ ID NO: 324)where X₁ is any amino acid and X₃ is G or S.

In one aspect, the present disclosure provides an anti-PCSK9 Adnectincomprising an FG loop having the sequence EX₄X₁X₅X₁X₁X₆GYX₄HRP (SEQ IDNO: 325), where X₁ is any amino acid; X₄ is Y or F; X₅ is Y, F, or W;and X₆ is S or A.

In one aspect, the present disclosure provides an anti-PCSK9 Adnectincomprising a BC loop having the sequence SW(X₁)_(Z)X₂G (SEQ ID NO: 323),a DE loop having the sequence PX₁X₁X₁X₃T (SEQ ID NO: 324), and an FGloop having the sequence EX₄X₁X₅X₁X₁X₆GYX₄HRP (SEQ ID NO: 325) asdefined herein.

In one aspect, the present disclosure provides an anti-PCSK9 Adnectincomprising a BC loop having the sequence SWEPFSRLPGGGE (SEQ ID NO: 106),a DE loop having the sequence PX₁X₁X₁X₃T (SEQ ID NO: 324), and an FGloop having the sequence EX₄X₁X₅X₁X₁X₆GYX₄HRP (SEQ ID NO: 325) asdefined herein.

In one aspect, the present disclosure provides an anti-PCSK9 Adnectincomprising a BC loop having the sequence (X₁)_(Z)X₂G (SEQ ID NO: 449)where X₁ is any amino acid, Z is a number from 6-9, and X₂ is Y or H.

In one aspect, the present disclosure provides an anti-PCSK9 Adnectincomprising a DE loop having the sequence X₁X₁X₁X₃, (SEQ ID NO: 450)where X₁ is any amino acid and X₃ is G or S.

In one aspect, the present disclosure provides an anti-PCSK9 Adnectincomprising a BC loop having the sequence (X₁)_(Z)X₂G (SEQ ID NO: 449), aDE loop having the sequence X₁X₁X₁X₃ (SEQ ID NO: 450), and an FG loophaving the sequence EX₄X₁X₅X₁X₁X₆GYX₄HR (SEQ ID NO: 451) as definedherein.

In some embodiments, there is at least one amino acid deletion from theN-terminus of the PCSK9 Adnectin.

In some embodiments, there is at least one amino acid deletion,insertion or substitution from the C-terminus of the PCSK9 Adnectin.

In some embodiments, a linker is added to the C-terminus of the PCSK9Adnectin.

In some embodiments, the PCSK9 Adnectin can be conjugated to a non-¹⁰Fn3moiety such as Human Serum Albumin (HSA) as described in PCT PublicationNos. WO 2009/133208 and WO 2009/083804.

In some embodiments, the PCSK9 Adnectin may have mutations in the AB, CDand EF loop amino acid sequences as described in PCT Publication Nos. WO2009/133208 and WO 2009/083804.

In one aspect, the anti-PCSK9 Adnectin further comprises apharmacokinetic (PK) moiety. In one embodiment, the PK moiety comprisespolyethylene glycol (PEG). In certain embodiments, the PK moietycomprises an Fc region. In some embodiments, the PK comprises one ormore serum albumin-binding Adnectins. Exemplary anti-PCSK9 Adnectin-Fcfusions proteins are shown in Table 1. Exemplary anti-PCSK9-serumalbumin binding Adnectin comprise SEQ ID NO: 618 or 619.

In certain embodiments, an anti-PCSK9 Adnectin having a PK moietycomprises the sequence as set forth in SEQ ID NO: 322.

In another aspect, the anti-PCSK9 Adnectin does not comprise any PKmoiety (i.e., a “naked” anti-PCSK9 Adnectin). In certain embodiments,the naked anti-PCSK9 Adnectin may be administered at a frequency thatcan sufficiently achieve the desired therapeutic effect. In anotherembodiment, the naked anti-PCSK9 Adnectin can be administered using anextended release formulation (e.g., subcutaneous formulation). In someembodiments, the extended release formulation increases the length ofthe absorption phase, or extends that pharmacodynamic effect, or both.Simply to illustrate, an extended release formulation comprises apropylene glycol/PBS solution.

In one aspect, the application provides an anti-PCSK9 Adnectin useful inthe treatment of atherosclerosis, hypercholesterolemia and othercholesterol related diseases.

In one aspect, the present invention provides a fusion polypeptidecomprising a fibronectin type III tenth (¹⁰Fn3) domain that binds toserum albumin and an anti-PCSK9 Adnectin, wherein the serum albuminbinding ¹⁰Fn3 domain binds to serum albumin, e.g., HSA, with a Kd of 1uM or less. In certain embodiments, the ¹⁰Fn3 domain that binds to serumalbumin comprises an amino acid sequence at least 70% identical to SEQID NO: 330. In one embodiment, the ¹⁰Fn3 domain that binds to serumalbumin comprises a BC loop having the amino acid sequence set forth inSEQ ID NO: 331, a DE loop having the amino acid sequence set forth inSEQ ID NO: 332, and an FG loop having the amino acid sequence set forthin SEQ ID NO:333. In another embodiment, the ¹⁰Fn3 domain that binds toserum albumin comprises one or more of a BC loop having the amino acidsequence set forth in SEQ ID NO: 331, a DE loop having the amino acidsequence set forth in SEQ ID NO: 332, and an FG loop having the aminoacid sequence set forth in SEQ ID NO: 333.

In one embodiment, the serum albumin binding ¹⁰Fn3 domain of the fusionpolypeptide also binds to one or more of rhesus serum albumin (RhSA),cynomolgus monkey serum albumin (CySA), or murine serum albumin (MuSA).In other embodiments, the ¹⁰Fn3 domain that binds to serum albumin doesnot cross-react with one or more of RhSA, CySA or MuSA.

In certain embodiments, the serum albumin binding ¹⁰Fn3 domain of thefusion polypeptide binds to HSA with a Kd of 1 uM or less. In someembodiments, the serum albumin binding ¹⁰Fn3 domain binds to HSA with aKd of 500 nM or less. In other embodiments, the serum albumin binding¹⁰Fn3 domain binds to HSA with a Kd of at least 200 nM, 100 nM, 50 nM,20 nM, 10 nM, or 5 nM.

In other embodiments, the serum albumin binding ¹⁰Fn3 domain of thefusion polypeptide binds to domain I or II of HSA. In one embodiment,the serum albumin binding ¹⁰Fn3 domain binds to both domains I and II ofHSA. In some embodiments, the serum albumin binding ¹⁰Fn3 domain bindsto HSA at a pH range of 5.5 to 7.4. In other embodiments, the serumalbumin binding ¹⁰Fn3 domain binds to HSA with a Kd of 200 nM or less atpH 5.5. In another embodiment, the serum albumin binding ¹⁰Fn3 domainbinds to HSA with a Kd of at least 500 nM, 200 nM, 100 nM, 50 nM, 20 nM,10 nM, or 5 nM at a pH range of 5.5 to 7.4. In one embodiment, the serumalbumin binding ¹⁰Fn3 domain binds to HSA with a Kd of at least 500 nM,200 nM, 100 nM, 50 nM, 20 nM, 10 nM, or 5 nM at pH 5.5.

In some embodiments, the serum half-life of the fusion polypeptide inthe presence of serum albumin is at least 5-fold greater than the serumhalf-life of the fusion polypeptide in the absence of serum albumin. Incertain embodiments, the serum half-life of the fusion polypeptide inthe presence of serum albumin is at least 2-fold, 5-fold, 7-fold,10-fold, 12-fold, 15-fold, 20-fold, 22-fold, 25-fold, 27-fold, or30-fold greater than the serum half-life of the fusion polypeptide inthe absence of serum albumin. In some embodiments, the serum albumin isany one of HSA, RhSA, CySA, or MuSA.

In certain embodiments, the serum half-life of the fusion polypeptide inthe presence of serum albumin is at least 20 hours. In certainembodiments, the serum half-life of the fusion polypeptide in thepresence of serum albumin is at least 10 hours, 12 hours, 15 hours, 20hours, 25 hours, 30 hours, 40 hours, 50 hours, 75 hours, 90 hours, 100hours, 110 hours, 120 hours, 130 hours, 150 hours, 170 hours, or 200hours. In some embodiments, the half-life of the fusion polypeptide isobserved in a primate (e.g., human or monkey) or a mouse.

In any of the foregoing aspects and embodiments, the ¹⁰Fn3 domain thatbinds serum albumin comprises a sequence selected from SEQ ID NO: 334,338, 342, 346, and 348-370.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows an alignment of exemplary anti-PCSK9 Adnectin amino acidsequences. The BC, DE and FG loop amino acid sequences are identified byunderlining, italics/underlining or bold/underlining, respectively.

FIG. 2 is a schematic depicting the PCSK9: Epidermal Growth Factorprecursor homology domain (EGFA domain) fluorescence resonance energytransfer (FRET) assay which was used to measure potency of PCSK9:LDLRinhibiting PCSK9 Adnectins as described in Example 2.

FIG. 3 shows the curve generated from a FRET assay which was used tomeasure the inhibition of human PCSK9:EGFA by PCSK9 Adnectin clones1459D05, 1784F03, 1813E02, 1922G04 and 1923B02 (panel A) and clones1459D05, 2012A04, 2011H05 and 2013E01 (panel B) as described in Example2.

FIG. 4 shows the curve generated from a FRET assay which was used tomeasure the inhibition of human PCSK9:ATI000972 interaction by PCSK9Adnectin clones 1459D05, 1784F03, 1813E02, 1922G04 and 1923B02 (panel A)and clones 2011H05, 2012A04 and 2013E01 (panel B) as described inExample 2.

FIG. 5 shows the activity of the PCSK9 Adnectin clones 1459D05, 1784F03,1813E02, 1922G04 and 1923B02 (panel A) and clones 2011H05, 2012A04 and2013E01 (panel B) in the direct binding human PCSK9 FRET assay asdescribed in Example 2.

FIG. 6 shows the inhibition of PCSK9 activity in HepG2 cells assayed bythe DiI-LDL uptake method as described in Example 2.

FIG. 7 shows the inhibition of PCSK9-induced LDLR depletion from HepG2cell surface by PCSK9 Adnectin clones 1459D05, 1784F03, 2012A04, and2011H05 (panel A) and Clone ID 2011H05, 2012A04 and 2013E01 (panel B) asdescribed in Example 2. The EC₅₀ (nM) of 1784F03, 2012A04, 2011H05, and2013E01 are 15.98, 7.78, 8.85, and 12.41, respectively; the percentageof PCSK9 inhibition at 75 nM of PCSK9 Adnectin clones 1459D05, 1784F03,2012A04, 2011H05, and 2013E01 are 66.8, 150.2, 190.1, 177.4, and 152.2,respectively.

FIG. 8 shows the in vivo effect of PCSK9 Adnectin ATI000959 (100 mg/kg)on plasma cholesterol (panel A) and plasma unbound hPCSK9 (panel B) inhPCSK9 overexpressing transgenic mice as described in Example 3.ATI000959 contains a 40 kDa branched NOF PEG.

FIG. 9 shows the in vivo effect of the PCSK9 Adnectin ATI001114 (10 or60 mg/kg) on plasma cholesterol levels (panel A) and on plasma unboundhPCSK9 levels (panel B) in hPCSK9 overexpressing transgenic mice asdescribed in Example 3.

FIG. 10 shows the in vivo effect of the PCSK9 Adnectins ATI000959 (panelA) or ATI001114 (panel B) administered at 5 mg/kg intraperitoneal(i.p.), single dose, on unbound plasma hPCSK9 in normal expresser hPCSK9transgenic mice (mean+/−SD) as described in Example 3.

FIG. 11 shows the dose-dependent effect of the PCSK9 Adnectin ATI001114on unbound hPCSK9 in normal expresser hPCSK9 transgenic mice asdescribed in Example 3.

FIG. 12 shows the effect of single dose of the PCSK9 Adnectin ATI001114(5 mg/kg i.v.) on LDL-C lowering in cynomolgus monkeys (mean+/−SEM, n=3)as described in Example 3.

FIG. 13. ITC determination of PCSK9 Adnectin affinity and stoichiometryof binding to hPCSK9. PCSK9 Adnectins bind hPCSK9 with 1:1stoichiometry. The left panel shows data for PCSK9 Adnectin ATI001081;the right panel shows data for PCSK9 Adnectin ATI001174.

FIG. 14. Inhibition of PCSK9:EGFA FRET assay (left panel) andPCSK9:ATI-972 FRET assay (right panel) by PCSK9 Adnectins.

FIG. 15. Inhibition of PCSK9-induced LDLR depletion from HepG2 cellsurface by anti-PCSK9 Adnectins.

FIG. 16. Inhibition of PCSK9-AF647 cell entry in HepG2 cells.

FIG. 17. Plasma unbound hPCSK9 levels in transgenic mice treated withPRD460 (dosed i.p.).

FIG. 18. Effect of PRD460 (15 mg/kg i.v.) on LDL-C and free PCSK9 incyno monkeys (mean+/−SEM, n=3).

FIG. 19. Effect of ATI-1081 (also referred to as ATI001081) on unboundPCSK9 levels in cynomolgus monkeys.

FIG. 20. Effect of ATI-1081 on LDL-C levels in cynomolgus monkeys.

FIG. 21. Effect of ATI-1081 in PBS vehicle in transgenic mice. Thefigure illustrates level of unbound plasma hPCSK9 in transgenic mice.

FIG. 22. Effect of ATI-1081 dosed subcutaneously in PG vehicle intransgenic mice. The figure illustrates level of unbound hPCSK9 intransgenic mice.

FIG. 23. In vivo HSA half-life in mice. HSA was injected into nude miceat 20 mg/kg (panel A) or 50 mg/kg (panel B).

FIG. 24. Half-life determination of SABA 1.1 (panel A), SABA2.1 (panelB), SABA3.1 (panel C), and SABA4.1 (panel D) in mice.

FIG. 25. Graph showing summary of half-life enhancement in mice ofSABA1-4 when co-injected with HSA.

FIG. 26. Half-life determination for SABA1.1 (panel A) and SABA5.1(panel B) in cynomolgus monkey.

FIG. 27. SABA1.2 binding to albumins from human, mouse and rat by directbinding ELISA assay.

FIG. 28. Determination of SABA1.1 and HSA stoichiometry. SABA1.1 and HSAbind with a stoichiometry of 1:1.

FIG. 29. BIACORE® analysis of SABA1.2 binding to recombinant domainfragments of HSA.

FIG. 30. Pharmacokinetic profile for SABA1.2 in cynomolgus monkeys dosedat 1 mpk and 10 mpk.

FIG. 31. Pharmacokinetic profile for SABA1.2 in monkeys dosedintravenously or subcutaneously at 1 mpk.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

By a “polypeptide” is meant any sequence of two or more amino acids,regardless of length, post-translation modification, or function.“Polypeptide,” “peptide,” and “protein” are used interchangeably herein.Polypeptides can include natural amino acids and non-natural amino acidssuch as those described in U.S. Pat. No. 6,559,126. Polypeptides canalso be modified in any of a variety of standard chemical ways (e.g., anamino acid can be modified with a protecting group; the carboxy-terminalamino acid can be made into a terminal amide group; the amino-terminalresidue can be modified with groups to, e.g., enhance lipophilicity; orthe polypeptide can be chemically glycosylated or otherwise modified toincrease stability or in vivo half-life). Polypeptide modifications caninclude the attachment of another structure such as a cyclic compound orother molecule to the polypeptide and can also include polypeptides thatcontain one or more amino acids in an altered configuration (i.e., R orS; or, L or D). The peptides of the invention are proteins derived fromthe tenth type III domain of fibronectin that have been modified to bindspecifically to PCSK9 and are referred to herein as, “anti-PCSK9Adnectin” or “PCSK9 Adnectin”.

The term “PK” is an acronym for “pharmacokinetic” and encompassesproperties of a compound including, by way of example, absorption,distribution, metabolism, and elimination by a subject. A “PK modulationprotein” or “PK moiety” refers to any protein, peptide, or moiety thataffects the pharmacokinetic properties of a biologically active moleculewhen fused to or administered together with the biologically activemolecule. Examples of a PK modulation protein or PK moiety include PEG,human serum albumin (HSA) binders (as disclosed in U.S. Publication Nos.2005/0287153 and 2007/0003549, PCT Publication Nos. WO 2009/083804 andWO 2009/133208, and SABA molecules as described herein), human serumalbumin, Fc or Fc fragments and variants thereof, and sugars (e.g.,sialic acid).

“Percent (%) amino acid sequence identity” herein is defined as thepercentage of amino acid residues in a candidate sequence that areidentical with the amino acid residues in a selected sequence, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. Alignmentfor purposes of determining percent amino acid sequence identity can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR®) software. Those skilledin the art can determine appropriate parameters for measuring alignment,including any algorithms needed to achieve maximal alignment over thefull-length of the sequences being compared.

An “isolated” polypeptide is one that has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials thatwould interfere with diagnostic or therapeutic uses for the polypeptide,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the polypeptide willbe purified (1) to greater than 95% by weight of polypeptide asdetermined by the Lowry method, and most preferably more than 99% byweight, (2) to a degree sufficient to obtain at least residues ofN-terminal or internal amino acid sequence by use of a spinning cupsequenator, or (3) to homogeneity by SDS-PAGE under reducing ornonreducing condition using Coomassie blue or, preferably, silver stain.Isolated polypeptide includes the polypeptide in situ within recombinantcells since at least one component of the polypeptide's naturalenvironment will not be present. Ordinarily, however, isolatedpolypeptide will be prepared by at least one purification step.

The notations “mpk”, “mg/kg”, or “mg per kg” refer to milligrams perkilogram. All notations are used interchangeably throughout the presentdisclosure.

The “half-life” of an amino acid sequence or compound can generally bedefined as the time taken for the serum concentration of the polypeptideto be reduced by 50%, in vivo, for example due to degradation of thesequence or compound and/or clearance or sequestration of the sequenceor compound by natural mechanisms. The half-life can be determined inany manner known per se, such as by pharmacokinetic analysis. Suitabletechniques will be clear to the person skilled in the art, and may forexample generally involve the steps of suitably administering to theprimate a suitable dose of the amino acid sequence or compound of theinvention; collecting blood samples or other samples from said primateat regular intervals; determining the level or concentration of theamino acid sequence or compound of the invention in said blood sample;and calculating, from (a plot of) the data thus obtained, the time untilthe level or concentration of the amino acid sequence or compound of theinvention has been reduced by 50% compared to the initial level upondosing. Reference is, for example, made to the standard handbooks, suchas Kenneth, A. et al., Chemical Stability of Pharmaceuticals: A Handbookfor Pharmacists and in Peters et al., Pharmacokinete Analysis: APractical Approach (1996). Reference is also made to Gibaldi, M. et al.,Pharmacokinetics, 2nd Rev. Edition, Marcel Dekker (1982).

Half-life can be expressed using parameters such as the t_(1/2)-alpha,t_(1/2)-beta, HL_Lambda_z, and the area under the curve (AUC). In thepresent specification, an “increase in half-life” refers to an increasein any one of these parameters, any two of these parameters, any threeof these parameters or all four of these parameters. An “increase inhalf-life” in particular refers to an increase in the t_(1/2)-betaand/or HL_Lambda_z, either with or without an increase in thet_(1/2)-alpha and/or the AUC or both.

Overview

This application provides Adnectins against human PCSK9. In order toidentify PCSK9 specific antagonists, PCSK9 was presented to largesynthetic libraries of Adnectins. Adnectins that bound to PCSK9 werescreened for PCSK9 binding, for biophysical properties, and for PCSK9inhibitory activity. The anti-PCSK9 Adnectins were mutated and subjectedto further selective pressure by lowering the target concentration andselecting for anti-PCSK9 Adnectins with slow off-rates. From thisoptimization process, a family of Adnectins was identified as PCSK9specific inhibitors with favorable biochemical and biophysicalproperties.

Fibronectin Based Scaffolds

One aspect of the application provides for polypeptides comprising Fn3domain in which one or more of the solvent accessible loops has beenrandomized or mutated. The Fn3 domain is an Fn3 domain derived from thewild-type tenth module of the human fibronectin type III domain (¹⁰Fn3):VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT (SEQ ID NO: 1). In the ¹⁰Fn3sequence, the BC, DE and FG loops are underlined.

As described herein, non-ligand binding sequences of ¹⁰Fn3, i.e., the“¹⁰Fn3 scaffold”, may be altered provided that the ¹⁰Fn3 retains ligandbinding function and/or structural stability. A variety of mutant ¹⁰Fn3scaffolds have been reported. In one aspect, one or more of Asp 7, Glu9, and Asp 23 is replaced by another amino acid, such as, for example, anon-negatively charged amino acid residue (e.g., Asn, Lys, etc.). Thesemutations have been reported to have the effect of promoting greaterstability of the mutant ¹⁰Fn3 at neutral pH as compared to the wild-typeform (See, PCT Publication No. WO 02/04523). A variety of additionalalterations in the ¹⁰Fn3 scaffold that are either beneficial or neutralhave been disclosed. See, for example, Batori et al., Protein Eng.,15(12):1015-1020 (December 2002); Koide et al., Biochemistry,40(34):10326-10333 (Aug. 28, 2001).

Both variant and wild-type ¹⁰Fn3 proteins are characterized by the samestructure, namely seven beta-strand domain sequences designated Athrough G and six loop regions (AB loop, BC loop, CD loop, DE loop, EFloop, and FG loop) which connect the seven beta-strand domain sequences.The beta strands positioned closest to the N- and C-termini may adopt abeta-like conformation in solution. In SEQ ID NO: 1, the AB loopcorresponds to residues 15-16, the BC loop corresponds to residues21-30, the CD loop corresponds to residues 39-45, the DE loopcorresponds to residues 51-56, the EF loop corresponds to residues60-66, and the FG loop corresponds to residues 76-87 (Xu et al.,Chemistry & Biology, 9:933-942 (2002)).

In some embodiments, the ¹⁰Fn3 polypeptide may be at least 40%, 50%,60%, 65%, 70%, 75%, 80%, 85%, or 90% identical to the human ¹⁰Fn3domain, shown in SEQ ID NO: 1. Much of the variability will generallyoccur in one or more of the loops. Each of the beta or beta-like strandsof a ¹⁰Fn3 polypeptide may consist essentially of an amino acid sequencethat is at least 80%, 85%, 90%, 95% or 100% identical to the sequence ofa corresponding beta or beta-like strand of SEQ ID NO: 1, provided thatsuch variation does not disrupt the stability of the polypeptide inphysiological conditions.

The disclosure provides polypeptides comprising a tenth fibronectin typeIII (¹⁰Fn3) domain, wherein the ¹⁰Fn3 domain comprises a loop, AB; aloop, BC; a loop, CD; a loop, DE; a loop EF; and a loop FG; and has atleast one loop selected from loop BC, DE, and FG with an altered aminoacid sequence relative to the sequence of the corresponding loop of thehuman ¹⁰Fn3 domain. In some embodiments, the BC and FG loops arealtered, and in some embodiments, the BC, DE, and FG loops are altered,i.e., the Fn3 domains comprise non-naturally occurring loops. In someembodiments, the AB, CD and/or the EF loops are altered. By “altered” ismeant one or more amino acid sequence alterations relative to a templatesequence (corresponding human fibronectin domain) and includes aminoacid additions, deletions, and substitutions. Altering an amino acidsequence may be accomplished through intentional, blind, or spontaneoussequence variation, generally of a nucleic acid coding sequence, and mayoccur by any technique, for example, PCR, error-prone PCR, or chemicalDNA synthesis.

In some embodiments, one or more loops selected from BC, DE, and FG maybe extended or shortened in length relative to the corresponding humanfibronectin loop. In some embodiments, the length of the loop may beextended by 2-25 amino acids. In some embodiments, the length of theloop may be decreased by 1-11 amino acids. To optimize antigen binding,therefore, the length of a loop of ¹⁰Fn3 may be altered in length aswell as in sequence to obtain the greatest possible flexibility andaffinity in antigen binding.

In some embodiments, the polypeptide comprises a Fn3 domain thatcomprises an amino acid sequence at least 80, 85, 90, 95, 98, 99 or 100%identical to the non-loop regions of SEQ ID NO: 1, wherein at least oneloop selected from BC, DE, and FG is altered. In some embodiments, thealtered BC loop has up to 10 amino acid substitutions, up to 4 aminoacid deletions, up to 10 amino acid insertions, or a combinationthereof. In some embodiments, the altered DE loop has up to 6 amino acidsubstitutions, up to 4 amino acid deletions, up to 13 amino acidinsertions or a combination thereof. In some embodiments, the FG loophas up to 12 amino acid substitutions, up to 11 amino acid deletions, upto 25 amino acid insertions or a combination thereof.

As described above, amino acid residues corresponding to residues 21-30,51-56, and 76-87 of SEQ ID NO: 1 define the BC, DE, and FG loops,respectively. However, it should be understood that not every residuewithin the loop region needs to be modified in order to achieve a ¹⁰Fn3binder having strong affinity for a desired target (e.g., PCSK9).

For example, residues 21 (S) and 22 (W) of the BC loop as shown in SEQID NO: 1 do not need to be modified for binding PCSK9. That is, ¹⁰Fn3domains with high affinity binding to PCSK9 may be obtained by modifyingonly residues 23-30 of loop BC as shown in SEQ ID NO: 1. This isdemonstrated in the BC loops exemplified in Table 3, which indicatesthat only the residues spanning the italicized positions were altered.Therefore, in some embodiments, a BC loop according to this designationcomprises the italicized portion of any one of SEQ ID NOs: 2-17,106-135, and 301-303 as shown in Table 3. For example, in oneembodiment, a BC loop may comprise the sequence PPPSHGYG (residues 3-10of SEQ ID NO: 2), DAPAHAYG (residues 3-10 of SEQ ID NO: 5), EPFSRLPGGGE(residues 3-13 of SEQ ID NO: 106), or DAPADGGYG (residues 3-11 of SEQ IDNO: 107).

Similarly, positions 51 (P) and 56 (T) of loop DE as shown in SEQ ID NO:1 do not need to be modified for binding PCSK9. That is, ¹⁰Fn3 domainswith high affinity binding to PCSK9 may be obtained by modifying onlyresidues 52-55 of loop DE as shown in SEQ ID NO: 1. This is demonstratedin the DE loops exemplified in Table 3, which indicates that only theresidues spanning the italicized positions were altered. Therefore, insome embodiments, a DE loop according to this designation comprises theitalicized portion of any one of SEQ ID NOs: 18-27 and 136-141, as shownin Table 3. For example, in one embodiment, a DE loop may comprise thesequence PGKG (residues 2-5 of SEQ ID NO: 18), VGVG (residues 2-5 SEQ IDNO: 27), or VSKS (residues 2-5 of SEQ ID NO: 137).

Likewise, position 87 (P) of the FG loop as shown in SEQ ID NO: 1 doesnot need to be modified for binding PCSK9. That is, ¹⁰Fn3 domains withhigh affinity binding to PCSK9 may be obtained by modifying onlyresidues 76-86 of the FG loop as shown in SEQ ID NO: 1. This isdemonstrated in the FG loops exemplified in Table 3, which indicatesthat only the residues spanning the italicized positions were altered.Therefore, in some embodiments, an FG loop according to this designationcomprises the italicized portion of any one of SEQ ID NOs: 28-38 and142-172, as shown in Table 3. For example, in one embodiment, an FG loopmay comprise the sequence EYPYKHSGYYHR (residues 1-12 in SEQ ID NO: 28),EYPYDYSGYYHR (residues 1-12 in SEQ ID NO: 142), or EFDFVGAGYYHR(residues 1-12 in SEQ ID NO: 167).

In some embodiments, the present application demonstrates that the BC,DE, and FG loop regions can be generally described according toconsensus sequences. For example, the BC loop can be generally definedby the consensus sequence SW(X₁)_(Z)X₂G (SEQ ID NO: 323) where X₁ is anyamino acid, Z is a number from 6-9, and X₂ is Y or H. This consensussequence is exemplified by BC loops shown in Table 3 except for the BCloop defined by SEQ ID NO: 106. In other embodiments, Z is a numberselected from 2-5. In certain embodiments, Z is a number selected from10-15. In some embodiments, X₂ is any aromatic residue (i.e., Y, F, W,or H).

In another embodiment, the DE loop can be generally defined by theconsensus sequence PX₁X₁X₁X₃T, (SEQ ID NO: 324) where X₁ is any aminoacid and X₃ is G or S. This consensus is exemplified by the DE loopsshown in Table 3.

In another embodiment, the FG loop can be generally defined by theconsensus sequence EX₄X₁X₅X₁X₁X₆GYX₄HRP (SEQ ID NO: 325), where X₁ isany amino acid; X₄ is Y or F; X₅ is Y, F, or W; and X₆ is S or A. Thisconsensus is exemplified by the FG loops shown in Table 3.

Accordingly, in certain embodiments, the present disclosure provides aPCSK9 binding Adnectin comprising a BC loop having the sequenceSW(X₁)_(Z)X₂G (SEQ ID NO: 323), a DE loop having the sequence PX₁X₁X₁X₃T(SEQ ID NO: 324), and an FG loop having the sequenceEX₄X₁X₅X₁X₁X₆GYX₄HRP (SEQ ID NO: 325), as defined above.

In another embodiment, the present disclosure provides a PCSK9 bindingAdnectin comprising a BC loop having the sequence SWEPFSRLPGGGE (SEQ IDNO: 106), a DE loop having the sequence PX₁X₁X₁X₃T (SEQ ID NO: 324), andan FG loop having the sequence EX₄X₁X₅X₁X₁X₆GYX₄HRP (SEQ ID NO: 325), asdefined above.

In certain embodiments, a BC loop can be generally defined by theconsensus sequence (X₁)_(Z)X₂G (SEQ ID NO: 449) where X₁ is any aminoacid, Z is a number from 6-9, and X₂ is Y or H. This consensus sequenceis exemplified by BC loops shown in Table 3 except for the BC loopdefined by SEQ ID NO: 106. In other embodiments, Z is a number selectedfrom 2-5. In certain embodiments, Z is a number selected from 10-15. Insome embodiments, X₂ is any aromatic residue (i.e., Y, F, W, or H).

In certain embodiments, the DE loop can be generally defined by theconsensus sequence X₁X₁X₁X₃, (SEQ ID NO: 450) where X₁ is any amino acidand X₃ is G or S. This consensus is demonstrated by the DE loops shownin Table 3.

The FG loop is defined by the consensus sequence EX₄X₁X₅X₁X₁X₆GYX₄HR(SEQ ID NO: 451), where X₁ is any amino acid; X₄ is Y or F; X₅ is Y, F,or W; and X₆ is S or A. This consensus is exemplified by the FG loopsshown in Table 3.

Accordingly, in one embodiment, the present disclosure provides a PCSK9binding Adnectin having a BC loop comprising the sequence (X₁)_(Z)X₂G(SEQ ID NO: 449), a DE loop comprising the sequence X₁X₁X₁X₃ (SEQ ID NO:450), and an FG loop comprising the sequence EX₄X₁X₅X₁X₁X₆GYX₄HR (SEQ IDNO: 451), as defined above.

In certain embodiments, antibody-like proteins based on the ¹⁰Fn3scaffold can be defined generally by the following sequence:

(SEQ ID NO: 328)EVVAAT(X)_(a)SLLI(X)_(x)YYRITYGE(X)_(b)QEFTV(X)_(y)ATI(X)_(c)DYTITVYAV(X)_(z)ISINYRT

In SEQ ID NO:328, the AB loop is represented by X_(a), the CD loop isrepresented by X_(b), the EF loop is represented by X_(c), the BC loopis represented by X_(x), the DE loop is represented by X_(y), and the FGloop is represented by X_(z). X represents any amino acid and thesubscript following the X represents an integer of the number of aminoacids. In particular, a may be anywhere from 1-15, 2-15, 1-10, 2-10,1-8, 2-8, 1-5, 2-5, 1-4, 2-4, 1-3, 2-3, or 1-2 amino acids; and b, c, x,y and z may each independently be anywhere from 2-20, 2-15, 2-10, 2-8,5-20, 5-15, 5-10, 5-8, 6-20, 6-15, 6-10, 6-8, 2-7, 5-7, or 6-7 aminoacids. In preferred embodiments, a is 2 amino acids, b is 7 amino acids,c is 7 amino acids, x is 9 amino acids, y is 6 amino acids, and z is 12amino acids. The sequences of the beta strands may have anywhere from 0to 10, from 0 to 8, from 0 to 6, from 0 to 5, from 0 to 4, from 0 to 3,from 0 to 2, or from 0 to 1 substitutions, deletions or additions acrossall 7 scaffold regions relative to the corresponding amino acids shownin SEQ ID NO: 1. In an exemplary embodiment, the sequences of the betastrands may have anywhere from 0 to 10, from 0 to 8, from 0 to 6, from 0to 5, from 0 to 4, from 0 to 3, from 0 to 2, or from 0 to 1 conservativesubstitutions across all 7 scaffold regions relative to thecorresponding amino acids shown in SEQ ID NO: 1. In certain embodiments,the core amino acid residues are fixed and any substitutions,conservative substitutions, deletions or additions occur at residuesother than the core amino acid residues.

In exemplary embodiments, the BC, DE, and FG loops as represented by(X)_(x), (X)_(y), and (X)_(z), respectively, are replaced withpolypeptides comprising the BC, DE and FG loop sequences from any of thePCSK9 binders shown in Table 3, or the italicized portions thereof, orthe consensus sequences 323-325 or 449-451.

In certain embodiments, Antibody-like proteins based on the ¹⁰Fn3scaffold can be defined generally by the sequence:

(SEQ ID NO: 329)EVVAATPTSLLI(X)_(x)YYRITYGETGGNSPVQEFTV(X)_(y)ATISGLKPGVDYTITVYAV(X)_(z)ISINYRT

In SEQ ID NO:329, the BC loop is represented by X_(x), the DE loop isrepresented by X_(y), and the FG loop is represented by X_(z). Xrepresents any amino acid and the subscript following the X representsan integer of the number of amino acids. In particular, x, y and z mayeach independently be anywhere from 2-20, 2-15, 2-10, 2-8, 5-20, 5-15,5-10, 5-8, 6-20, 6-15, 6-10, 6-8, 2-7, 5-7, or 6-7 amino acids. Inpreferred embodiments, x is 9 amino acids, y is 6 amino acids, and z is12 amino acids. The sequences of the beta strands may have anywhere from0 to 10, from 0 to 8, from 0 to 6, from 0 to 5, from 0 to 4, from 0 to3, from 0 to 2, or from 0 to 1 substitutions, deletions or additionsacross all 7 scaffold regions relative to the corresponding amino acidsshown in SEQ ID NO: 1. In an exemplary embodiment, the sequences of thebeta strands may have anywhere from 0 to 10, from 0 to 8, from 0 to 6,from 0 to 5, from 0 to 4, from 0 to 3, from 0 to 2, or from 0 to 1conservative substitutions across all 7 scaffold regions relative to thecorresponding amino acids shown in SEQ ID NO: 1. In certain embodiments,the core amino acid residues are fixed and any substitutions,conservative substitutions, deletions or additions occur at residuesother than the core amino acid residues. In exemplary embodiments, theBC, DE, and FG loops as represented by (X)_(x), (X)_(y), and (X)_(z),respectively, are replaced with polypeptides comprising the BC, DE andFG loop sequences from any of the PCSK9 binders shown in Table 3, or theitalicized portions thereof, or the consensus sequences 323-325 or449-451.

In certain embodiments, an anti-PCSK9 Adnectin described herein maycomprise the sequence as set forth in SEQ ID NO: 328 or 329, wherein theBC, DE, and FG loops as represented by (X)_(x), (X)_(y), and (X)_(z),respectively, are replaced with a respective set of specified BC, DE,and FG loops from any of the clones in Table 3, or sequences at least75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the BC, DE or FGloop sequences of the clones listed in Table 3. In exemplaryembodiments, an anti-PCSK9 Adnectin as described herein is defined bySEQ ID NO: 329 and has a respective set of BC, DE and FG loop sequencesfrom any of the clones listed in Table 3. For example, clone 1459D05 inTable 3 comprises BC, DE, and FG loops as set forth in SEQ ID NOs: 2,18, and 28, respectively. Therefore, an anti-PCSK9 Adnectin based onthese loops may comprise SEQ ID NO: 328 or 329, wherein (X)_(x)comprises SEQ ID NO: 2, (X)_(y) comprises SEQ ID NO: 18, and (X)_(z)comprises SEQ ID NO: 28. Similar constructs are contemplated utilizingthe set of BC, DE and FG loops from the other clones in Table 3, or theconsensus sequences 323-325 or 449-451. The scaffold regions of suchanti-PCSK9 Adnectin may comprise anywhere from 0 to 20, from 0 to 15,from 0 to 10, from 0 to 8, from 0 to 6, from 0 to 5, from 0 to 4, from 0to 3, from 0 to 2, or from 0 to 1 substitutions, conservativesubstitutions, deletions or additions relative to the scaffold aminoacids residues of SEQ ID NO: 1. Such scaffold modifications may be made,so long as the anti-PCSK9 Adnectin is capable of binding PCSK9 with adesired K_(D).

In some embodiments, the BC loop of the protein of the inventioncomprises an amino acid sequence selected from the group consisting ofSWPPPSHGYG (SEQ ID NO: 2), SWRPPIHAYG (SEQ ID NO: 3), SWDAPIHAYG (SEQ IDNO:4), SWDAPAHAYG (SEQ ID NO:5) and SWDAPAVTYG (SEQ ID NO:6), SWSPPANGYG(SEQ ID NO:7), SWTPPPKGYG (SEQ ID NO:8), SWRPPSHAYG (SEQ ID NO:9),SWDPPSHAYG (SEQ ID NO: 10), SWEPPSHAYG (SEQ ID NO: 11), SWSPPSHAYG (SEQID NO: 12), SWRPPSNGHG (SEQ ID NO: 13), SWVPPSDDYG (SEQ ID NO: 14),SWVPSSHAYG (SEQ ID NO:15), SWDPSSHAYG (SEQ ID NO:16), and SWEPSSHAYG(SEQ ID NO:17). In further embodiments, the BC loop of the protein ofthe invention comprises an amino acid sequence selected from SEQ ID NOs:106-135 and 301-303. In other embodiments, the BC loop of the protein ofthe invention comprises the italicized portion of any one of SEQ ID NOs:2-17, 106-135, and 301-303 as shown in Table 3. For example, in oneembodiment, a BC loop comprises the sequence PPPSHGYG (residues 3-10 ofSEQ ID NO: 2), DAPAHAYG (residues 3-10 of SEQ ID NO: 5), EPFSRLPGGGE(residues 3-13 of SEQ ID NO: 106), or DAPADGGYG (residues 3-11 of SEQ IDNO: 107).

In some embodiments, the DE loop of the protein of the inventioncomprises an amino acid sequence selected from the group consisting ofPPGKGT (SEQ ID NO: 18), PIVEGT (SEQ ID NO:19), PGSEGT (SEQ ID NO:20),PGSKGT (SEQ ID NO:21), PGSKST (SEQ ID NO:22), PVGRGT (SEQ ID NO:23),PVGEGT (SEQ ID NO:24), PIGKGT (SEQ ID NO:25), PVNEGT (SEQ ID NO:26), andPVGVGT (SEQ ID NO:27). In further embodiments, the DE loop of theprotein of the invention comprises an amino acid sequence selected fromSEQ ID NOs: 136-141. In other embodiments, the DE loop of the protein ofthe invention comprises the italicized portion of any one of SEQ ID NOs:18-27 and 136-141 as shown in Table 3. For example, in one embodiment, aDE loop comprises the sequence PGKG (residues 2-5 of SEQ ID NO: 18),VGVG (residues 2-5 SEQ ID NO: 27), or VSKS (residues 2-5 of SEQ ID NO:137).

In some embodiments, the FG loop of the protein of the inventioncomprises an amino acid sequence selected from the group consisting ofEYPYKHSGYYHRP (SEQ ID NO:28), EYTFKHSGYYHRP (SEQ ID NO:29),EYTYKGSGYYHRP (SEQ ID NO:30), EYTYNGAGYYHRP (SEQ ID NO:31),EYTYIGAGYYHRP (SEQ ID NO:32), EYTYEGAGYYHRP (SEQ ID NO:33),EYAYNGAGYYHRP (SEQ ID NO:34), EYPWKGSGYYHRP (SEQ ID NO:35),EFPFKWSGYYHRP (SEQ ID NO:36), EFPWPHAGYYHRP (SEQ ID NO:37) andEYAFEGAGYYHRP (SEQ ID NO:38). In further embodiments, the FG loop of theprotein of the invention comprises an amino acid sequence selected fromSEQ ID NOs: 142-172. In other embodiments, the FG loop of the protein ofthe invention comprises the italicized portion of any one of SEQ ID NOs:28-38 and 142-172 as shown in Table 3. For example, in one embodiment,an FG loop comprises the sequence EYPYKHSGYYHR (residues 1-12 in SEQ IDNO: 28), EYPYDYSGYYHR (residues 1-12 in SEQ ID NO: 142), or EFDFVGAGYYHR(residues 1-12 in SEQ ID NO: 167).

In some embodiments, the protein of the invention comprises one BC loopsequence selected from the BC loop sequences having SEQ ID NOs: 2-17,106-135, and 301-303, or the italicized portion of any one of SEQ IDNOS: 2-17, 106-135, and 301-303, as shown in Table 3; one DE loopsequence selected from the DE loop sequences having SEQ ID NOs:18-27 and136-141, or the italicized portion of any one of SEQ ID NOS:18-27 and136-141 as shown in Table 3; and one FG loop sequence selected from theFG loop sequences having SEQ ID NOS: 28-38 and 142-172, or theitalicized portion of any one of SEQ ID NOS: 28-38 and 142-172 as shownin Table 3. In some embodiments, the protein of the invention comprisesa BC, DE and FG loop amino acid sequence at least 70, 75, 80, 85, 90,95, 98, 99 or 100% identical to of any one of SEQ ID NOS: 2-38, 106-172,301-303. In other embodiments, the protein of the invention comprises aBC, DE and FG loop amino acid sequence at least 70, 75, 80, 85, 90, 95,98, 99 or 100% identical to the italicized portion of any one of SEQ IDNOS: 2-38, 106-172, 301-303 as shown in Table 3.

In some embodiments, the anti-PCSK9 Adnectin comprises the amino acidsequence of any one of SEQ ID NOS: 39-76, 173-290, and 304-309. In someembodiments, the anti-PCSK9 Adnectin comprises the Fn3 domain amino acidsequence from position 3-96 of any one of SEQ ID NOS: 39-76, 173-290,and 304-309. In some embodiments, the anti-PCSK9 Adnectin comprises anamino acid sequence at least 70, 75, 80, 85, 90, 95, 98, 99 or 100%identical to any one of SEQ ID NOS:39-76, 173-290, and 304-309.

Fibronectin naturally binds certain types of integrins through itsintegrin-binding motif, “arginine-glycine-aspartic acid” (RGD). In someembodiments, the polypeptide comprises a ¹⁰Fn3 domain that lacks the(RGD) integrin binding motif. The integrin binding domain may be removedby altering the RGD sequence by amino acid substitution, deletion orinsertion.

In certain embodiments, the anti-PCSK9 Adnectin molecules of the presentinvention may be modified to comprise an N-terminal extension sequenceand/or a C-terminal extension. For example, an MG sequence may be placedat the N-terminus of the ¹⁰Fn3 defined by SEQ ID NO: 1. The M willusually be cleaved off, leaving a G at the N-terminus. Alternatively,the first 10 amino acids of the anti-PCSK9 Adnectins shown in Table 4may be replaced with an alternative N-terminal sequence, referred toherein as N-terminal extensions,

as shown in Table 6 (i.e., SEQ ID NOs: 371-379). In addition, an M, G orMG may also be placed N-terminal to any of the N-terminal extensionshaving SEQ ID NOs: 371-379. The anti-PCSK9 Adnectins described hereinmay also comprise alternative C-terminal tail sequences, referred toherein as C-terminal extension sequences. For example, the anti-PCSK9Adnectin sequences shown in Table 4 may be truncated at the threoninecorresponding to T94 of SEQ ID NO: 1 (i.e., truncated after the INYRT(SEQ ID NO: 636) portion of the sequence). Such truncated version may beused as therapeutic molecules in the truncated form, or alternativeC-terminal extensions may be added after the threonine residue.Exemplary C-terminal extension sequences are shown in Table 6 as SEQ IDNOs: 380-395. Exemplary anti-PCSK9 Adnectins comprising C-terminalextension sequences are shown in Table 4. For example, SEQ ID NO: 49(clone 1813E02) comprises the naturally occurring C-terminal extensionEIDKPSQ (SEQ ID NO: 380) followed by a His6 tag (SEQ ID NO: 637).However, it should be understood that the His6 tag is completelyoptional.

In certain embodiments, the C-terminal extension sequences (also called“tails”), comprise E and D residues, and may be between 8 and 50, 10 and30, 10 and 20, 5 and 10, and 2 and 4 amino acids in length. In someembodiments, tail sequences include ED-based linkers in which thesequence comprises tandem repeats of ED. In exemplary embodiments, thetail sequence comprises 2-10, 2-7, 2-5, 3-10, 3-7, 3-5, 3, 4 or 5 EDrepeats. In certain embodiments, the ED-based tail sequences may alsoinclude additional amino acid residues, such as, for example: EI (SEQ IDNO: 385), EID, ES, EC, EGS, and EGC. Such sequences are based, in part,on known Adnectin tail sequences, such as EIDKPSQ (SEQ ID NO: 380), inwhich residues D and K have been removed. In exemplary embodiments, theED-based tail comprises an E, I or EI (SEQ ID NO: 385) residues beforethe ED repeats.

In other embodiments, the N- or C-terminal sequences may be combinedwith other known linker sequences (e.g., SEQ ID NO: 396-419 in Table 6)as necessary when designing an anti-PCSK9 Adnectin fusion molecule.Exemplary anti-PCSK9 Adnectin comprising linker sequences are shown inTable 4 (e.g., SEQ ID NOs: 53, 55, and 57). In some embodiments,sequences may be placed at the C-terminus of the ¹⁰Fn3 domain tofacilitate attachment of a pharmacokinetic moiety. For example, acysteine containing linker such as GSGC (SEQ ID NO:77) may be added tothe C-terminus to facilitate site directed PEGylation on the cysteineresidue.

Pharmacokinetic Moieties

In one aspect, the application provides for anti-PCSK9 Adnectins furthercomprising a pharmacokinetic (PK) moiety. Improved pharmacokinetics maybe assessed according to the perceived therapeutic need. Often it isdesirable to increase bioavailability and/or increase the time betweendoses, possibly by increasing the time that a protein remains availablein the serum after dosing. In some instances, it is desirable to improvethe continuity of the serum concentration of the protein over time(e.g., decrease the difference in serum concentration of the proteinshortly after administration and shortly before the nextadministration). The anti-PCSK9 Adnectin may be attached to a moietythat reduces the clearance rate of the polypeptide in a mammal (e.g.,mouse, rat, or human) by greater than three-fold relative to theunmodified anti-PCSK9 Adnectin. Other measures of improvedpharmacokinetics may include serum half-life, which is often dividedinto an alpha phase and a beta phase. Either or both phases may beimproved significantly by addition of an appropriate moiety.

Moieties that tend to slow clearance of a protein from the blood, hereinreferred to as “PK moieties”, include polyoxyalkylene moieties, e.g.,polyethylene glycol, sugars (e.g., sialic acid), and well-toleratedprotein moieties (e.g., Fc and fragments and variants thereof,transferrin, or serum albumin). The anti-PCSK9 Adnectin may be fused toalbumin or a fragment (portion) or variant of albumin as described inU.S. Publication No. 2007/0048282. In some embodiments, the PCSK9Adnectin may be fused to one or more serum albumin binding Adnectin, asdescribed herein.

In some embodiments, the PK moiety is a serum albumin binding proteinsuch as those described in U.S. Publication Nos. 2007/0178082 and2007/0269422.

In some embodiments, the PK moiety is a serum immunoglobulin bindingprotein such as those described in U.S. Publication No. 2007/0178082

In some embodiments, the anti-PCSK9 Adnectin comprises polyethyleneglycol (PEG). One or more PEG molecules may be attached at differentpositions on the protein, and such attachment may be achieved byreaction with amines, thiols or other suitable reactive groups. Theamine moiety may be, for example, a primary amine found at theN-terminus of a polypeptide or an amine group present in an amino acid,such as lysine or arginine. In some embodiments, the PEG moiety isattached at a position on the polypeptide selected from the groupconsisting of: a) the N-terminus; b) between the N-terminus and the mostN-terminal beta strand or beta-like strand; c) a loop positioned on aface of the polypeptide opposite the target-binding site; d) between theC-terminus and the most C-terminal beta strand or beta-like strand; ande) at the C-terminus.

Pegylation may be achieved by site-directed pegylation, wherein asuitable reactive group is introduced into the protein to create a sitewhere pegylation preferentially occurs. In some embodiments, the proteinis modified to introduce a cysteine residue at a desired position,permitting site directed pegylation on the cysteine. PEG may vary widelyin molecular weight and may be branched or linear. In one embodiment thePEG has two branches. In another embodiment the PEG has four branches.

In some embodiments, the anti-PCSK9 Adnectin is fused to animmunoglobulin Fc domain, or a fragment or variant thereof. In anexemplary embodiment, the Fc domain is derived from an IgG1 subclass,however, other subclasses (e.g., IgG2, IgG3, and IgG4) may also be used.Shown below is the sequence of a human IgG1 immunoglobulin Fc domain,and the relative position of each region within the Fc domain areindicated based on the EU numbering format:

(SEQ ID NO: 315) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKThe core hinge sequence is underlined, and the CH1 region is italicized;the CH2 and CH3 regions are in regular text. It should be understoodthat the C-terminal lysine is optional.

The fusion may be formed by attaching an anti-PCSK9 Adnectin to eitherend of the Fc molecule, i.e., Fc-anti-PCSK9 Adnectin or anti-PCSK9Adnectin-Fc arrangements. In certain embodiments, the Fc and anti-PCSK9Adnectin are fused via a linker. Exemplary linker sequences includeAGGGGSG (SEQ ID NO: 310), GSGSGSGSGSGS (SEQ ID NO: 311), QPDEPGGS (SEQID NO: 312), ELQLEESAAEAQDGELD (SEQ ID NO: 313), TVAAPS (SEQ ID NO:314), KAGGGGSG (SEQ ID NO: 620), KGSGSGSGSGSGS (SEQ ID NO: 621),KQPDEPGGS (SEQ ID NO: 622), KELQLEESAAEAQDGELD (SEQ ID NO: 623), KTVAAPS(SEQ ID NO: 624), KAGGGGSGG (SEQ ID NO: 625), KGSGSGSGSGSGSG (SEQ ID NO:626), KQPDEPGGSG (SEQ ID NO: 627), KELQLEESAAEAQDGELDG (SEQ ID NO: 628),KTVAAPSG (SEQ ID NO: 629) AGGGGSGG (SEQ ID NO: 630), GSGSGSGSGSGSG (SEQID NO: 631), QPDEPGGSG (SEQ ID NO: 632), ELQLEESAAEAQDGELDG (SEQ ID NO:633), and TVAAPSG (SEQ ID NO: 634).

In some embodiments, the Fc region used in the anti-PCSK9 Adnectinfusion comprises the hinge region of an Fc molecule. As used herein, the“hinge” region comprises the core hinge residues spanning positions104-119 of SEQ ID NO: 315 (DKTHTCPPCPAPELLG; SEQ ID NO: 316) of IgG1,which corresponds to positions 221-236 according to EU numbering. Incertain embodiments, the anti-PCSK9 Adnectin-Fc fusion adopts amultimeric structure (e.g., dimer) owing, in part, to the cysteineresidues at positions 109 and 112 of SEQ ID NO: 315 (EU numbering 226and 229, respectively) within the hinge region. In other embodiments,the hinge region as used herein, may further include residues derivedfrom the CH1 and CH2 regions that flank the core hinge sequence, asshown in SEQ ID NO: 315.

In some embodiments, the hinge sequence may include substitutions thatconfer desirable pharmacokinetic, biophysical, and/or biologicalproperties. Some exemplary hinge sequences include

(SEQ ID NO: 317 EPKSSDKTHTCPPCPAPELLGGPS;core hinge region underlined)

(SEQ ID NO: 318 EPKSSDKTHTCPPCPAPELLGGSS;core hinge region underlined),

(SEQ ID NO: 319 EPKSSGSTHTCPPCPAPELLGGSS;core hinge region underlined),

(SEQ ID NO: 320 DKTHTCPPCPAPELLGGPS;core hinge region underlined), and

(SEQ ID NO: 321 DKTHTCPPCPAPELLGGSS,core hinge region underlined). In one embodiment, the residue P atposition 122 (EU numbering 238) of SEQ ID NO: 315 has been replaced withS to ablate Fc effector function; this replacement is exemplified inhinges having any one of SEQ ID NOs: 318, 319, and 321. In anotherembodiment, the residues DK at positions 104-105 of SEQ ID NO: 315 (EUnumbering 221-222) have been replaced with GS to remove a potential clipsite; this replacement is exemplified in SEQ ID NO: 319. In anotherembodiment, the C at position 103 of SEQ ID NO: 315 (EU numbering 220)has been replaced with S to prevent improper cystine bond formation inthe absence of a light chain; this replacement is exemplified in SEQ IDNOs: 317-319.

In certain embodiments, an antiPCSK9 Adnectin-Fc fusion may have thefollowing configurations: 1) anti-PCSK9 Adnectin-hinge-Fc or 2)hinge-Fc-anti-PCSK9 Adnectin. Therefore, any anti-PCSK9 Adnectin of thepresent invention can be fused to an Fc region comprising a hingesequence according to these configurations. In some embodiments, alinker may be used to join the anti-PCSK9 Adnectin to the hinge-Fcmoiety, for example, an exemplary fusion protein may have theconfiguration hinge-anti-PCSK9 Adnectin-linker-Fc. Additionally,depending on the system in which the fusion polypeptide is produced, aleader sequence may placed at the N-terminus of the fusion polypeptide.For example, if the fusion is produced in a mammalian system, a leadersequence such as METDTLLLWVLLLWVPGSTG (SEQ ID NO: 326) may be added tothe N-terminus of the fusion molecule. If the fusion is produced in E.coli, the fusion sequence will be preceded by a methionine.

The following sequence exemplifies an anti-PCSK9 Adnectin-hinge-Fcconstruct produced in a mammalian system:

(SEQ ID NO: 322) METDTLLLWVLLLWVPGSTG GVSDVPRDLEVVAATPTSLLISWVPPSDDYGYYRITYGETGGNSPVQEFTVPIGKGTATISGLKPGVDYTITVYAVEFP WPHAGYYHRPISINYRTEIEPKSSGSTHTCPPCPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK.Here, the Fc domain comprises the human IgG1 CH2 and CH3 regions asfollows: VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 448) and the hingesequence of SEQ ID NO:319. In SEQ ID NO: 322, the leader sequence is inbold, the anti-PCSK9 Adnectin sequence is in italics, and the hingeregion is underlined. It should be understood that the lysine at the endof SEQ ID NO: 322 is optional. The efficacy of the polypeptide fusion asset forth in SEQ ID NO: 322 (also described herein as PRD460) isdemonstrated in Example 4.

Exemplary PCSK9 Adnectin-Fc fusions are shown in Table 1. All sequencesmay begin with a methionine or a mammalian leader sequence (e.g., SEQ IDNO: 326).

TABLE 1 Exemplary Anti-PCSK9 Adnectin-Fc Fusion Proteins SEQ Clone or IDName Description Sequence PCSK9 Adnectin-X₁-Fc C-Terminal Fusions 4521459D05-Fc X1 is optional and when GVSDVPRDLEVVAATPTSLLISWPPPSHG fusionpresent can be selected from YGYYRITYGETGGNSPVQEFTVPPGKGTA E, EI, EID,EIDK (SEQ ID TISGLKPGVDYTITVYAVEYPYKHSGYYH NO: 384), EIE, and EIEKRPISINYRT-X₁-X₂- (SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSHselected from hinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs:317-321; STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 453 1784F03-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWRPPIHA fusion present can be selected fromYGYYRITYGETGGNSPVQEFTVPIVEGTA E, EI, EID, EIDK (SEQ IDTISGLKPGVDYTITVYAVEYTFKHSGYYH NO: 384), EIE, and EIEK RPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 454 1784F03-m1- X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWDAPIHA Fc fusion present can be selectedfrom YGYYRITYGETGGNSPVQEFTVPGSEGTA E, EI, EID, EIDK (SEQ IDTISGLKPGVDYTITVYAVEYTFKHSGYYH NO: 384), EIE, and EIEK RPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 455 1784F03-m2- X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWDAPAHA Fc fusion present can be selectedfrom YGYYRITYGETGGNSPVQEFTVPGSKGTA E, EI, EID, EIDK (SEQ IDTISGLKPGVDYTITVYAVEYTFKHSGYYH NO: 384), EIE, and EIEK RPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 456 1784F03-m3- X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWDAPAVT Fc fusion present can be selectedfrom YGYYRITYGETGGNSPVQEFTVPGSKSTA E, EI, EID, EIDK (SEQ IDTISGLKPGVDYTITVYAVEYTFKHSGYYH NO: 384), EIE, and EIEK RPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 457 1813E02-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWSPPANG fusion present can be selected fromYGYYRITYGETGGNSPVQEFTVPVGRGTA E, EI, EID, EIDK (SEQ IDTISGLKPGVDYTITVYAVEYTYKGSGYYH NO: 384), EIE, and EIEK RPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 458 1923B02-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWTPPPKG fusion present can be selected fromYGYYRITYGETGGNSPVQEFTVPVGEGTA E, EI, EID, EIDK (SEQ IDTISGLKPGVDYTITVYAVEYTYNGAGYYH NO: 384), EIE, and EIEK RPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK 459 1923B02(N82I)- X1 is optionaland when GVSDVPRDLEVVAATPTSLLISWTPPPKG Fc fusion present can be selectedfrom YGYYRITYGETGGNSPVQEFTVPVGEGTA E, EI, EID, EIDK (SEQ IDTISGLKPGVDYTITVYAVEYTYIGAGYYH NO: 384), EIE, and EIEK RPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 460 1923B02(N82E)- X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWTPPPKG Fc fusion present can be selectedfrom YGYYRITYGETGGNSPVQEFTVPVGEGTA E, EI, EID, EIDK (SEQ IDTISGLKPGVDYTITVYAVEYTYEGAGYYH NO: 384), EIE, and EIEK RPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 461 1923B02(T80A)- X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWTPPPKG Fc fusion present can be selectedfrom YGYYRITYGETGGNSPVQEFTVPVGEGTA E, EI, EID, EIDK (SEQ IDTISGLKPGVDYTITVYAVEYAYNGAGYYH NO: 384), EIE, and EIEK RPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 462 1922G04-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWRPPSHA fusion present can be selected fromYGYYRITYGETGGNSPVQEFTVPIGKGTA E, EI, EID, EIDK (SEQ IDTISGLKPGVDYTITVYAVEYPWKGSGYYH NO: 384), EIE, and EIEK RPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 463 1922G04(R25D)- X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWDPPSHA Fc fusion present can be selectedfrom YGYYRITYGETGGNSPVQEFTVPIGKGTA E, EI, EID, EIDK (SEQ IDTISGLKPGVDYTITVYAVEYPWKGSGYYH NO: 384), EIE, and EIEK RPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 464 1922G04(R25E)- X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWEPPSHA Fc fusion present can be selectedfrom YGYYRITYGETGGNSPVQEFTVPIGKGTA E, EI, EID, EIDK (SEQ IDTISGLKPGVDYTITVYAVEYPWKGSGYYH NO: 384), EIE, and EIEK RPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 465 1922G04(R25S)- X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWSPPSHA Fc fusion present can be selectedfrom YGYYRITYGETGGNSPVQEFTVPIGKGTA E, EI, EID, EIDK (SEQ IDTISGLKPGVDYTITVYAVEYPWKGSGYYH NO: 384), EIE, and EIEK RPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 466 2012A04-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWRPPSNG fusion present can be selected fromHGYYRITYGETGGNSPVQEFTVPVNEGTA E, EI, EID, EIDK (SEQ IDTISGLKPGVDYTITVYAVEFPFKWSGYYH NO: 384), EIE, and EIEK RPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 467 2013E01-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWVPPSDD fusion present can be selected fromYGYYRITYGETGGNSPVQEFTVPIGKGTA E, EI, EID, EIDK (SEQ IDTISGLKPGVDYTITVYAVEFPWPHAGYYH NO: 384), EIE, and EIEK RPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 468 2011H05-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWVPSSHA fusion present can be selected fromYGYYRITYGETGGNSPVQEFTVPVGVGTA E, EI, EID, EIDK (SEQ IDTISGLKPGVDYTITVYAVEYAFEGAGYYH NO: 384), EIE, and EIEK RPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 469 2011H05(V23D)- X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWDPSSHA Fc fusion present can be selectedfrom YGYYRITYGETGGNSPVQEFTVPVGVGTA E, EI, EID, EIDK (SEQ IDTISGLKPGVDYTITVYAVEYAFEGAGYYH NO: 384), EIE, and EIEK RPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 470 2011H05(V23E)- X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWEPSSHA Fc fusion present can be selectedfrom YGYYRITYGETGGNSPVQEFTVPVGVGTA E, EI, EID, EIDK (SEQ IDTISGLKPGVDYTITVYAVEYAFEGAGYYH NO: 384), EIE, and EIEK RPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 471 2381B02-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWEPFSRL fusion present can be selected fromPGGGEYYRITYGETGGNSPLQQFTVPGSK E, EI, EID, EIDK (SEQ IDGTATISGLKPGVDYTITVYAVEYPYDYSG NO: 384), EIE, and EIEKYYHRPISINYRT-X₁-X₂- (SEQ ID NO: 635); X2 isVFLFPPKPKDTLMISRTPEVTCVVVDVSH selected from hingeEDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 472 2381B04-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWEPFSRL fusion present can be selected fromPGGGEYYRITYGETGGNSPLQQFTVPGSK E, EI, EID, EIDK (SEQ IDGTATISGLKPGVDYTITVYAVEYPYEHSG NO: 384), EIE, and EIEKYYHRPISINYRT-X₁-X₂- (SEQ ID NO: 635); X2 isVFLFPPKPKDTLMISRTPEVTCVVVDVSH selected from hingeEDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 473 2381B06-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWEPFSRL fusion present can be selected fromPGGGEYYRITYGETGGNSPLQQFTVPGSK E, EI, EID, EIDK (SEQ IDGTATISGLKPGVDYTITVYAVEYPYPHSG NO: 384), EIE, and EIEKYYHRPISINYRT-X₁-X₂- (SEQ ID NO: 635); X2 isVFLFPPKPKDTLMISRTPEVTCVVVDVSH selected from hingeEDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 474 2381B08-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWDAPADG fusion present can be selected fromGYGYYRITYGETGGNSPVQEFTVPSSKGT E, EI, EID, EIDK (SEQ IDATISGLKPGVDYTITVYAVEYTFPGAGYY NO: 384), EIE, and EIEK HRPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 475 2381D02-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWEPFSRL fusion present can be selected fromPGGGEYYRITYGETGGNSPLQQFTVPGSK E, EI, EID, EIDK (SEQ IDGTATISGLKPGVDYTITVYAVEYPYDHSG NO: 384), EIE, and EIEKYYHRPISINYRT-X₁-X₂- (SEQ ID NO: 635); X2 isVFLFPPKPKDTLMISRTPEVTCVVVDVSH selected from hingeEDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 476 2381D04-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWEPFSRL fusion present can be selected fromPGGGEYYRITYGETGGNSPLQQFTVPGSK E, EI, EID, EIDK (SEQ IDGTATISGLKPGVDYTITVYAVEFPYDHSG NO: 384), EIE, and EIEKYYHRPISINYRT-X₁-X₂- (SEQ ID NO: 635); X2 isVFLFPPKPKDTLMISRTPEVTCVVVDVSH selected from hingeEDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 477 2381F11-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWDAPADG fusion present can be selected fromGYGYYRITYGETGGNSPVQEFTVPVSKST E, EI, EID, EIDK (SEQ IDATISGLKPGVDYTITVYAVEYTFPGAGYY NO: 384), EIE, and EIEK HRPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 478 2381G03-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWEPFSRL fusion present can be selected fromPGGGEYYRITYGETGGNSPLQQFTVPGSK E, EI, EID, EIDK (SEQ IDGTATISGLKPGVDYTITVYAVEFPYAHSG NO: 384), EIE, and EIEKYYHRPISINYRT-X₁-X₂- (SEQ ID NO: 635); X2 isVFLFPPKPKDTLMISRTPEVTCVVVDVSH selected from hingeEDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 479 2381G09-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWDAPAGD fusion present can be selected fromGYGYYRITYGETGGNSPVQEFTVPVSKGT E, EI, EID, EIDK (SEQ IDATISGLKPGVDYTITVYAVEFTFPGAGYY NO: 384), EIE, and EIEK HRPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 480 2381H03-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWEPFSRL fusion present can be selected fromPGGGEYYRITYGETGGNSPLQQFTVPGSK E, EI, EID, EIDK (SEQ IDGTATISGLKPGVDYTITVYAVEYPYAHSG NO: 384), EIE, and EIEKYFHRPISINYRT-X₁-X₂- (SEQ ID NO: 635); X2 isVFLFPPKPKDTLMISRTPEVTCVVVDVSH selected from hingeEDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 481 2382A01-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWAAPAGG fusion present can be selected fromGYGYYRITYGETGGNSPVQEFTVPVSKGT E, EI, EID, EIDK (SEQ IDATISGLKPGVDYTITVYAVEYDFPGAGYY NO: 384), EIE, and EIEK HRPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 482 2382B10-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWDAPADA fusion present can be selected fromYGYYRITYGETGGNSPVQEFTVPSSKGTA E, EI, EID, EIDK (SEQ IDTISGLKPGVDYTITVYAVEYDFPGSGYYH NO: 384), EIE, and EIEK RPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 483 2382B09-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWDAPADA fusion present can be selected fromYGYYRITYGETGGNSPVQEFTVPVSKGTA E, EI, EID, EIDK (SEQ IDTISGLKPGVDYTITVYAVEFDYPGSGYYH NO: 384), EIE, and EIEK RPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 484 2382C05-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWDAPADG fusion present can be selected fromAYGYYRITYGETGGNSPVQEFTVPVSKGT E, EI, EID, EIDK (SEQ IDATISGLKPGVDYTITVYAVEYSFPGAGYY NO: 384), EIE, and EIEK HRPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 485 2382C09-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWDAPAEG fusion present can be selected fromYGYYRITYGETGGNSPVQEFTVPVSKGTA E, EI, EID, EIDK (SEQ IDTISGLKPGVDYTITVYAVEFDFPGSGYYH NO: 384), EIE, and EIEK RPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 486 2382D03-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWDAPADE fusion present can be selected fromAYGYYRITYGETGGNSPVQEFTVPVSKGT E, EI, EID, EIDK (SEQ IDATISGLKPGVDYTITVYAVEFDFPGAGYY NO: 384), EIE, and EIEK HRPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 487 2382D05-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWDAPADG fusion present can be selected fromGYGYYRITYGETGGNSPVQEFTVPVSKGT E, EI, EID, EIDK (SEQ IDATISGLKPGVDYTITVYAVEFDFPGAGYY NO: 384), EIE, and EIEK HRPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 488 2382D08-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWDAPADG fusion present can be selected fromYGYYRITYGETGGNSPVQEFTVPVSKGTA E, EI, EID, EIDK (SEQ IDTISGLKPGVDYTITVYAVEFPFPGSGYYH NO: 384), EIE, and EIEK RPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 489 2382D09-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWDAPAEG fusion present can be selected fromYGYYRITYGETGGNSPVQEFTVPVSKGTA E, EI, EID, EIDK (SEQ IDTISGLKPGVDYTITVYAVEFDFPGAGYYH NO: 384), EIE, and EIEK RPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 490 2382F02-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWDAPAGG fusion present can be selected fromGYGYYRITYGETGGNSPVQEFTVPVSKGT E, EI, EID, EIDK (SEQ IDATISGLKPGVDYTITVYAVEFDFPGSGYY NO: 384), EIE, and EIEK HRPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 491 2382F03-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWDAPAAD fusion present can be selected fromAYGYYRITYGETGGNSPVQEFTVPVSKGT E, EI, EID, EIDK (SEQ IDATISGLKPGVDYTITVYAVEFNFPGAGYY NO: 384), EIE, and EIEK HRPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 492 2382F05-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWDAPAEA fusion present can be selected fromGKHYGYYRITYGETGGNSPVQEFTVPVSK E, EI, EID, EIDK (SEQ IDGTATISGLKPGVDYTITVYAVEFDFPGAG NO: 384), EIE, and EIEKYYHRPISINYRT-X₁-X₂- (SEQ ID NO: 635); X2 isVFLFPPKPKDTLMISRTPEVTCVVVDVSH selected from hingeEDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 493 2382F08-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWDAPAEA fusion present can be selected fromYGYYRITYGETGGNSPVQEFTVPVSKGTA E, EI, EID, EIDK (SEQ IDTISGLKPGVDYTITVYAVEFTYPGSGYYH NO: 384), EIE, and EIEK RPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 494 2382F09-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWDAPAAA fusion present can be selected fromYGYYRITYGETGGNSPVQEFTVPVSKGTA E, EI, EID, EIDK (SEQ IDTISGLKPGVDYTITVYAVEYDFPGSGYYH NO: 384), EIE, and EIEK RPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 495 2382G04-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWDAPAGG fusion present can be selected fromGYGYYRITYGETGGNSPVQEFTVPSSKGT E, EI, EID, EIDK (SEQ IDATISGLKPGVDYTITVYAVEFDFPGAGYY NO: 384), EIE, and EIEK HRPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 496 2382H10-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWDAPAGG fusion present can be selected fromYGYYRITYGETGGNSPVQEFTVPVSKGTA E, EI, EID, EIDK (SEQ IDTISGLKPGVDYTITVYAVEFDFPGSGYYH NO: 384), EIE, and EIEK RPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 497 2382H11-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWDAPADG fusion present can be selected fromYGYYRITYGETGGNSPVQEFTVPVFKGTA E, EI, EID, EIDK (SEQ IDTISGLKPGVDYTITVYAVEFDYPGSGYYH NO: 384), EIE, and EIEK RPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 498 2382H04-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWDAPAAG fusion present can be selected fromGYGYYRITYGETGGNSPVQEFTVPSSKGT E, EI, EID, EIDK (SEQ IDATISGLKPGVDYTITVYAVEYDFPGAGYY NO: 384), EIE, and EIEK HRPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 499 2382H07-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWDAPADA fusion present can be selected fromYGYYRITYGETGGNSPVQEFTVPGSKGTA E, EI, EID, EIDK (SEQ IDTISGLKPGVDYTITVYAVEFDFPGSGYYH NO: 384), EIE, and EIEK RPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 500 2382H09-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWDAPAAA fusion present can be selected fromYGYYRITYGETGGNSPVQEFTVPSSKGTA E, EI, EID, EIDK (SEQ IDTISGLKPGVDYTITVYAVEFDFPGSGYYH NO: 384), EIE, and EIEK RPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 501 2451A02-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWDAPAAG fusion present can be selected fromYGYYRITYGETGGNSPVQEFTVPVSKGTA E, EI, EID, EIDK (SEQ IDTISGLKPGVDYTITVYAVEFPFPGSGYYH NO: 384), EIE, and EIEK RPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 502 2451B05-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWDAPAGG fusion present can be selected fromYGYYRITYGETGGNSPVQEFTVPSSKGTA E, EI, EID, EIDK (SEQ IDTISGLKPGVDYTITVYAVEFDYPGSGYYH NO: 384), EIE, and EIEK RPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 503 2451B06-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWDAPADG fusion present can be selected fromGYGYYRITYGETGGNSPVQEFTVPVSKGT (equivalent to E, EI, EID, EIDK (SEQ IDATISGLKPGVDYTITVYAVEFDFPGAGYY 2382D05) NO: 384), EIE, and EIEKHRPISINYRT-X₁-X₂- (SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSHselected from hinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs:317-321; STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 504 2451C06-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWDAPAGA fusion present can be selected fromASYGYYRITYGETGGNSPVQEFTVPVSKG E, EI, EID, EIDK (SEQ IDTATISGLKPGVDYTITVYAVEFPFPGAGY NO: 384), EIE, and EIEK YHRPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 505 2451D05-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWDAPAGA fusion present can be selected fromYGYYRITYGETGGNSPVQEFTVPVSKGTA E, EI, EID, EIDK (SEQ IDTISGLKPGVDYTITVYAVEFDFPGSGYYH NO: 384), EIE, and EIEK RPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 506 2451F03-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWDPPAEG fusion present can be selected fromYGYYRITYGETGGNSPVQEFTVPVSKGTA E, EI, EID, EIDK (SEQ IDTISGLKPGVDYTITVYAVEFNFPGSGYYH NO: 384), EIE, and EIEK RPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 507 2451G01-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWDAPAGG fusion present can be selected fromYGYYRITYGETGGNSPVQEFTVPSSKGTA E, EI, EID, EIDK (SEQ IDTISGLKPGVDYTITVYAVEFDFPGSGYYH NO: 384), EIE, and EIEK RPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 508 2451H07-Fc X1 is optional andwhen GITDVPRDLEVVAATPTSLLISWNPPDVN fusion present can be selected fromYGYYRITYGETGGNSPLQEFTVPVSKGTA E, EI, EID, EIDK (SEQ IDTISGLKPGVDYTITVYAVEYPYAHAGYYH NO: 384), EIE, and EIEK RPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 509 2382E03-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWDAPAGD fusion present can be selected fromGYGYYRITYGETGGNSPVQEFTVPVSKGT E, EI, EID, EIDK (SEQ IDATISGLKPGVDYTITVYAVEFDFPGAGYY NO: 384), EIE, and EIEK HRPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 510 2382E04-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWDAPAGG fusion present can be selected fromGYGYYRITYGETGGNSPVQEFTVPVSKGT E, EI, EID, EIDK (SEQ IDATISGLKPGVDYTITVYAVEFTFPGAGYY NO: 384), EIE, and EIEK HRPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 511 2382E05-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWDAPAEG fusion present can be selected fromGYGYYRITYGETGGNSPVQEFTVPVSKGT E, EI, EID, EIDK (SEQ IDATISGLKPGVDYTITVYAVEFDFPGAGYY NO: 384), EIE, and EIEK HRPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 512 2382E09-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWDAPAEA fusion present can be selected fromYGYYRITYGETGGNSPVQEFTVPVSKGTA E, EI, EID, EIDK (SEQ IDTISGLKPGVDYTITVYAVEYDFPGSGYYH NO: 384), EIE, and EIEK RPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 513 2381A04-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWEPFSRL fusion present can be selected fromPGGGEYYRITYGETGGNSPLQQFTVPGSK E, EI, EID, EIDK (SEQ IDGTATISGLKPGVDYTITVYAVEYPYPFSG NO: 384), EIE, and EIEKYYHRPISINYRT-X₁-X₂- (SEQ ID NO: 635); X2 isVFLFPPKPKDTLMISRTPEVTCVVVDVSH selected from hingeEDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 514 2381A08-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWDAPADG fusion present can be selected fromGYGYYRITYGETGGNSPVQEFTVPGSKGT E, EI, EID, EIDK (SEQ IDATISGLKPGVDYTITVYAVEYDFPGAGYY NO: 384), EIE, and EIEK HRPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 515 2381B10-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWDAPAGG fusion present can be selected fromGYGYYRITYGETGGNSPVQEFTVPVSKGT E, EI, EID, EIDK (SEQ IDATISGLKPGVDYTITVYAVEYNFIGAGYY NO: 384), EIE, and EIEK HRPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 516 2381C08-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWDAPADG fusion present can be selected fromAYGYYRITYGETGGNSPVQEFTVPVSKGT E, EI, EID, EIDK (SEQ IDATISGLKPGVDYTITVYAVEFPYPFAGYY NO: 384), EIE, and EIEK HRPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 517 2381G06-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWSEKLDG fusion present can be selected fromKARRGYYRITYGETGGNSPVQQFTVPGSK E, EI, EID, EIDK (SEQ IDGTATISGLKPGVDYTITVYAVEFPYDHSG NO: 384), EIE, and EIEKYYHRPISINYRT-X₁-X₂- (SEQ ID NO: 635); X2 isVFLFPPKPKDTLMISRTPEVTCVVVDVSH selected from hingeEDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 518 2381H01-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWSPRDST fusion present can be selected fromGLVRRGYYRITYGETGGNSPVQQFTVPGS E, EI, EID, EIDK (SEQ IDKGTATISGLKPGVDYTITVYAVEYPYDHS NO: 384), EIE, and EIEKGYYHRPISINYRT-X₁-X₂- (SEQ ID NO: 635); X2 isVFLFPPKPKDTLMISRTPEVTCVVVDVSH selected from hingeEDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 519 2381H06-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWGDVRTN fusion present can be selected fromEARQGYYRITYGETGGNSPLQGFTVPGSK E, EI, EID, EIDK (SEQ IDGTATISGLKPGVDYTITVYAVEYTYEHSG NO: 384), EIE, and EIEKYYHRPISINYRT-X₁-X₂- (SEQ ID NO: 635); X2 isVFLFPPKPKDTLMISRTPEVTCVVVDVSH selected from hingeEDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 520 2381H09-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWDAPAGG fusion present can be selected fromGYGYYRITYGETGGNSPVQEFTVPVSKGT E, EI, EID, EIDK (SEQ IDATISGLKPGVDYTITVYAVEFDFVGAGYY NO: 384), EIE, and EIEK HRPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 521 2382B11-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWDAPAAA fusion present can be selected fromYGYYRITYGETGGNSPVQEFTVPVSKGTA E, EI, EID, EIDK (SEQ IDTISGLKPGVDYTITVYAVEYDFAGSGYYH NO: 384), EIE, and EIEK RPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 522 2382B08-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWDAPADA fusion present can be selected fromYGYYRITYGETGGNSPVQEFTVPSSKGTA E, EI, EID, EIDK (SEQ IDTISGLKPGVDYTITVYAVEFAFPGAGYYH NO: 384), EIE, and EIEK RPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 523 2382C11-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWDAPAGG fusion present can be selected fromYGYYRITYGETGGNSPVQEFTVPVSKGTA E, EI, EID, EIDK (SEQ IDTISGLKPGVDYTITVYAVEYDFAGSGYYH NO: 384), EIE, and EIEK RPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 524 2382G03-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWDAPAEA fusion present can be selected fromEAYGYYRITYGETGGNSPVQEFTVPVSKG E, EI, EID, EIDK (SEQ IDTATISGLKPGVDYTITVYAVEYVFPGAGY NO: 384), EIE, and EIEK YHRPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 525 2382H03-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWDAPAEG fusion present can be selected fromAYGYYRITYGETGGNSPVQEFTVPVSKGT E, EI, EID, EIDK (SEQ IDATISGLKPGVDYTITVYAVEYPYPFAGYY NO: 384), EIE, and EIEK HRPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 526 2451A10-Fc X1 is optional andwhen GVTDVPRDMEVVAATPTSLLISWQPPAVT fusion present can be selected fromYGYYRITYGETGGNSTLQQFTVPVYKGTA E, EI, EID, EIDK (SEQ IDTISGLKPGVDYTITVYAVEYPYDHSGYYH NO: 384), EIE, and EIEK RPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 527 2451B02-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWDAPAAA fusion present can be selected fromYGYYRITYGETGGNSPVQEFTVPVSKGTA E, EI, EID, EIDK (SEQ IDTISGLKPGVDYTITVYAVEFDYPGSGYYH NO: 384), EIE, and EIEK RPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 528 2451C11-Fc X1 is optional andwhen GIVDVPRDLEVVAATPTSLLISWDPPAGA fusion present can be selected fromYGYYRITYGETGGNSPKQQFTVPGYKGTA E, EI, EID, EIDK (SEQ IDTISGLKPGVDYTITVYAVEYPYDHSGYYH NO: 384), EIE, and EIEK RPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK 529 2451H01-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWDAPAAG fusion present can be selected fromYGYYRITYGETGGNSPVQEFTVPVSKGTA E, EI, EID, EIDK (SEQ IDTISGLKPGVDYTITVYAVEYDFPGSGYYH NO: 384), EIE, and EIEK RPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 530 2011B11-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWAPPSDA fusion present can be selected fromYGYYRITYGETGGNSPVQEFTVPIGKGTA E, EI, EID, EIDK (SEQ IDTISGLKPGVDYTITVYAVEYPYSHAGYYH NO: 384), EIE, and EIEK RPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 531 2971A03-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWDPPSDD fusion present can be selected fromYGYYRITYGETGGNSPVQEFTVPIGKGTA E, EI, EID, EIDK (SEQ IDTISGLKPGVDYTITVYAVEFPWPHAGYYH NO: 384), EIE, and EIEK RPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 532 2971A09-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWDAPADD fusion present can be selected fromYGYYRITYGETGGNSPVQEFTVPIGKGTA E, EI, EID, EIDK (SEQ IDTISGLKPGVDYTITVYAVEFPWPHAGYYH NO: 384), EIE, and EIEK RPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 533 2971E02-Fc X1 is optional andwhen GVSDVPRDLEVVAATPTSLLISWDAPSDD fusion present can be selected fromYGYYRITYGETGGNSPVQEFTVPIGKGTA E, EI, EID, EIDK (SEQ IDTISGLKPGVDYTITVYAVEFPWPHAGYYH NO: 384), EIE, and EIEK RPISINYRT-X₁-X₂-(SEQ ID NO: 635); X2 is VFLFPPKPKDTLMISRTPEVTCVVVDVSH selected fromhinge EDPEVKFNWYVDGVEVHNAKTKPREEQYN sequences SEQ ID NOs: 317-321;STYRVVSVLTVLHQDWLNGKEYKCKVSNK the Fc may optionallyALPAPIEKTISKAKGQPREPQVYTLPPSR include a C-terminal KDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG X₁-Fc-X₂-PCSK9 Adnectin N-TerminalFusions 534 Fc-1459D05 X1 is selected from hinge X₁- fusion sequencesSEQ ID NOs: 317-321; VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWPPPSHGY GYYRITYGETGGNSPVQEFTVPPGKGTATISGLKPGVDYTITVYAVEYPYKHSGYYHR PISINYRT-X₃ 535 Fc-1784F03 X1 is selectedfrom hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWRPPIHAY GYYRITYGETGGNSPVQEFTVPIVEGTATISGLKPGVDYTITVYAVEYTFKHSGYYHR PISINYRT-X₃ 536 Fc-1784F03- X1 is selectedfrom hinge X₁- m1 fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWDAPIHAY GYYRITYGETGGNSPVQEFTVPGSEGTATISGLKPGVDYTITVYAVEYTFKHSGYYHR PISINYRT-X₃ 537 Fc-1784F03- X1 is selectedfrom hinge X₁- m2 fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWDAPAHAY GYYRITYGETGGNSPVQEFTVPGSKGTATISGLKPGVDYTITVYAVEYTFKHSGYYHR PISINYRT-X₃ 538 Fc-1784F03- X1 is selectedfrom hinge X₁- m3 fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWDAPAVTY GYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVEYTFKHSGYYHR PISINYRT-X₃ 539 Fc-1813E02 X1 is selectedfrom hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWSPPANGY GYYRITYGETGGNSPVQEFTVPVGRGTATISGLKPGVDYTITVYAVEYTYKGSGYYHR PISINYRT-X₃ 540 Fc-1923B02 X1 is selectedfrom hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWTPPPKGY GYYRITYGETGGNSPVQEFTVPVGEGTATISGLKPGVDYTITVYAVEYTYNGAGYYHR PISINYRT-X₃ 541 Fc- X1 is selected fromhinge X₁- 1923B02(N82I) sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH fusion X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWTPPPKGY GYYRITYGETGGNSPVQEFTVPVGEGTATISGLKPGVDYTITVYAVEYTYIGAGYYHR PISINYRT-X₃ 542 Fc- X1 is selected fromhinge X₁- 1923B02(N82E) sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH fusion X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWTPPPKGY GYYRITYGETGGNSPVQEFTVPVGEGTATISGLKPGVDYTITVYAVEYTYEGAGYYHR PISINYRT-X₃ 543 Fc- X1 is selected fromhinge X₁- 1923B02(T80A) sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH fusion X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWTPPPKGY GYYRITYGETGGNSPVQEFTVPVGEGTATISGLKPGVDYTITVYAVEYAYNGAGYYHR PISINYRT-X₃ 544 Fc-1922G04 X1 is selectedfrom hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWRPPSHAY GYYRITYGETGGNSPVQEFTVPIGKGTATISGLKPGVDYTITVYAVEYPWKGSGYYHR PISINYRT-X₃ 545 Fc- X1 is selected fromhinge X₁- 1922G04(R25D) sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH fusion X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWDPPSHAY GYYRITYGETGGNSPVQEFTVPIGKGTATISGLKPGVDYTITVYAVEYPWKGSGYYHR PISINYRT-X₃ 546 Fc- X1 is selected fromhinge X₁- 1922G04(R25E) sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH fusion X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWEPPSHAY GYYRITYGETGGNSPVQEFTVPIGKGTATISGLKPGVDYTITVYAVEYPWKGSGYYHR PISINYRT-X₃ 547 Fc- X1 is selected fromhinge X₁- 1922G04(R25S) sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH fusion X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWSPPSHAY GYYRITYGETGGNSPVQEFTVPIGKGTATISGLKPGVDYTITVYAVEYPWKGSGYYHR PISINYRT-X₃ 548 Fc-2012A04 X1 is selectedfrom hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWRPPSNGH GYYRITYGETGGNSPVQEFTVPVNEGTATISGLKPGVDYTITVYAVEFPFKWSGYYHR PISINYRT-X₃ 549 Fc-2013E01 X1 is selectedfrom hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWVPPSDDY GYYRITYGETGGNSPVQEFTVPIGKGTATISGLKPGVDYTITVYAVEFPWPHAGYYHR PISINYRT-X₃ 550 Fc-2011H05 X1 is selectedfrom hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWVPSSHAY GYYRITYGETGGNSPVQEFTVPVGVGTATISGLKPGVDYTITVYAVEYAFEGAGYYHR PISINYRT-X₃ 551 Fc- X1 is selected fromhinge X₁- 2011H05(V23D) sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH fusion X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWDPSSHAY GYYRITYGETGGNSPVQEFTVPVGVGTATISGLKPGVDYTITVYAVEYAFEGAGYYHR PISINYRT-X₃ 552 Fc- X1 is selected fromhinge X₁- 2011H05(V23E) sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH fusion X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWEPSSHAY GYYRITYGETGGNSPVQEFTVPVGVGTATISGLKPGVDYTITVYAVEYAFEGAGYYHR PISINYRT-X₃ 553 Fc-2381B02 X1 is selectedfrom hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWEPFSRLP GGGEYYRITYGETGGNSPLQQFTVPGSKGTATISGLKPGVDYTITVYAVEYPYDYSGY YHRPISINYRT-X₃ 554 Fc-2381B04 X1 isselected from hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWEPFSRLP GGGEYYRITYGETGGNSPLQQFTVPGSKGTATISGLKPGVDYTITVYAVEYPYEHSGY YHRPISINYRT-X₃ 555 Fc-2381B06 X1 isselected from hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWEPFSRLP GGGEYYRITYGETGGNSPLQQFTVPGSKGTATISGLKPGVDYTITVYAVEYPYPHSGY YHRPISINYRT-X₃ 556 Fc-2381B08 X1 isselected from hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWDAPADGG YGYYRITYGETGGNSPVQEFTVPSSKGTATISGLKPGVDYTITVYAVEYTFPGAGYYH RPISINYRT-X₃ 557 Fc-2381D02 X1 is selectedfrom hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWEPFSRLP GGGEYYRITYGETGGNSPLQQFTVPGSKGTATISGLKPGVDYTITVYAVEYPYDHSGY YHRPISINYRT-X₃ 558 Fc-2381D04 X1 isselected from hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWEPFSRLP GGGEYYRITYGETGGNSPLQQFTVPGSKGTATISGLKPGVDYTITVYAVEFPYDHSGY YHRPISINYRT-X₃ 559 Fc-2381F11 X1 isselected from hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWDAPADGG YGYYRITYGETGGNSPVQEFTVPVSKSTATISGLKPGVDYTITVYAVEYTFPGAGYYH RPISINYRT-X₃ 560 Fc-2381G03 X1 is selectedfrom hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWEPFSRLP GGGEYYRITYGETGGNSPLQQFTVPGSKGTATISGLKPGVDYTITVYAVEFPYAHSGY YHRPISINYRT-X₃ 561 Fc-2381G09 X1 isselected from hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWDAPAGDG YGYYRITYGETGGNSPVQEFTVPVSKGTATISGLKPGVDYTITVYAVEFTFPGAGYYH RPISINYRT-X₃ 562 Fc-2381H03 X1 is selectedfrom hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWEPFSRLP GGGEYYRITYGETGGNSPLQQFTVPGSKGTATISGLKPGVDYTITVYAVEYPYAHSGY FHRPISINYRT-X₃ 563 Fc-2382A01 X1 isselected from hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWAAPAGGG YGYYRITYGETGGNSPVQEFTVPVSKGTATISGLKPGVDYTITVYAVEYDFPGAGYYH RPISINYRT-X₃ 564 Fc-2382B10 X1 is selectedfrom hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWDAPADAY GYYRITYGETGGNSPVQEFTVPSSKGTATISGLKPGVDYTITVYAVEYDFPGSGYYHR PISINYRT-X₃ 565 Fc-2382B09 X1 is selectedfrom hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWDAPADAY GYYRITYGETGGNSPVQEFTVPVSKGTATISGLKPGVDYTITVYAVEFDYPGSGYYHR PISINYRT-X₃ 566 Fc-2382C05 X1 is selectedfrom hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWDAPADGA YGYYRITYGETGGNSPVQEFTVPVSKGTATISGLKPGVDYTITVYAVEYSFPGAGYYH RPISINYRT-X₃ 567 Fc-2382C09 X1 is selectedfrom hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWDAPAEGY GYYRITYGETGGNSPVQEFTVPVSKGTATISGLKPGVDYTITVYAVEFDFPGSGYYHR PISINYRT-X₃ 568 Fc-2382D03 X1 is selectedfrom hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWDAPADEA YGYYRITYGETGGNSPVQEFTVPVSKGTATISGLKPGVDYTITVYAVEFDFPGAGYYH RPISINYRT-X₃ 569 Fc-2382D05 X1 is selectedfrom hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWDAPADGG YGYYRITYGETGGNSPVQEFTVPVSKGTATISGLKPGVDYTITVYAVEFDFPGAGYYH RPISINYRT-X₃ 570 Fc-2382D08 X1 is selectedfrom hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWDAPADGY GYYRITYGETGGNSPVQEFTVPVSKGTATISGLKPGVDYTITVYAVEFPFPGSGYYHR PISINYRT-X₃ 571 Fc-2382D09 X1 is selectedfrom hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWDAPAEGY GYYRITYGETGGNSPVQEFTVPVSKGTATISGLKPGVDYTITVYAVEFDFPGAGYYHR PISINYRT-X₃ 572 Fc-2382F02 X1 is selectedfrom hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWDAPAGGG YGYYRITYGETGGNSPVQEFTVPVSKGTATISGLKPGVDYTITVYAVEFDFPGSGYYH RPISINYRT-X₃ 573 Fc-2382F03 X1 is selectedfrom hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWDAPAADA YGYYRITYGETGGNSPVQEFTVPVSKGTATISGLKPGVDYTITVYAVEFNFPGAGYYH RPISINYRT-X₃ 574 Fc-2382F05 X1 is selectedfrom hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWDAPAEAG KHYGYYRITYGETGGNSPVQEFTVPVSKGTATISGLKPGVDYTITVYAVEFDFPGAGY YHRPISINYRT-X₃ 575 Fc-2382F08 X1 isselected from hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWDAPAEAY GYYRITYGETGGNSPVQEFTVPVSKGTATISGLKPGVDYTITVYAVEFTYPGSGYYHR PISINYRT-X₃ 576 Fc-2382F09 X1 is selectedfrom hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWDAPAAAY GYYRITYGETGGNSPVQEFTVPVSKGTATISGLKPGVDYTITVYAVEYDFPGSGYYHR PISINYRT-X₃ 577 Fc-2382G04 X1 is selectedfrom hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWDAPAGGG YGYYRITYGETGGNSPVQEFTVPSSKGTATISGLKPGVDYTITVYAVEFDFPGAGYYH RPISINYRT-X₃ 578 Fc-2382H10 X1 is selectedfrom hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWDAPAGGY GYYRITYGETGGNSPVQEFTVPVSKGTATISGLKPGVDYTITVYAVEFDFPGSGYYHR PISINYRT-X₃ 579 Fc-2382H11 X1 is selectedfrom hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWDAPADGY GYYRITYGETGGNSPVQEFTVPVFKGTATISGLKPGVDYTITVYAVEFDYPGSGYYHR PISINYRT-X₃ 580 Fc-2382H04 X1 is selectedfrom hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWDAPAAGG YGYYRITYGETGGNSPVQEFTVPSSKGTATISGLKPGVDYTITVYAVEYDFPGAGYYH RPISINYRT-X₃ 581 Fc-2382H07 X1 is selectedfrom hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWDAPADAY GYYRITYGETGGNSPVQEFTVPGSKGTATISGLKPGVDYTITVYAVEFDFPGSGYYHR PISINYRT-X₃ 582 Fc-2382H09 X1 is selectedfrom hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWDAPAAAY GYYRITYGETGGNSPVQEFTVPSSKGTATISGLKPGVDYTITVYAVEFDFPGSGYYHR PISINYRT-X₃ 583 Fc-2451A02 X1 is selectedfrom hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWDAPAAGY GYYRITYGETGGNSPVQEFTVPVSKGTATISGLKPGVDYTITVYAVEFPFPGSGYYHR PISINYRT-X₃ 584 Fc-2451B05 X1 is selectedfrom hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWDAPAGGY GYYRITYGETGGNSPVQEFTVPSSKGTATISGLKPGVDYTITVYAVEFDYPGSGYYHR PISINYRT-X₃ 585 Fc-2451B06 X1 is selectedfrom hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH (equivalent to X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN 2382D05) linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWDAPADGG YGYYRITYGETGGNSPVQEFTVPVSKGTATISGLKPGVDYTITVYAVEFDFPGAGYYH RPISINYRT-X₃ 586 Fc-2451C06 X1 is selectedfrom hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWDAPAGAA SYGYYRITYGETGGNSPVQEFTVPVSKGTATISGLKPGVDYTITVYAVEFPFPGAGYY HRPISINYRT-X₃ 587 Fc-2451D05 X1 isselected from hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWDAPAGAY GYYRITYGETGGNSPVQEFTVPVSKGTATISGLKPGVDYTITVYAVEFDFPGSGYYHR PISINYRT-X₃ 588 Fc-2451F03 X1 is selectedfrom hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWDPPAEGY GYYRITYGETGGNSPVQEFTVPVSKGTATISGLKPGVDYTITVYAVEFNFPGSGYYHR PISINYRT-X₃ 589 Fc-2451G01 X1 is selectedfrom hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWDAPAGGY GYYRITYGETGGNSPVQEFTVPSSKGTATISGLKPGVDYTITVYAVEFDFPGSGYYHR PISINYRT-X₃ 590 Fc-2451H07 X1 is selectedfrom hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) ITDVPRDLEVVAATPTSLLISWNPPDVNY GYYRITYGETGGNSPLQEFTVPVSKGTATISGLKPGVDYTITVYAVEYPYAHAGYYHR PISINYRT-X₃ 591 Fc-2382E03 X1 is selectedfrom hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWDAPAGDG YGYYRITYGETGGNSPVQEFTVPVSKGTATISGLKPGVDYTITVYAVEFDFPGAGYYH RPISINYRT-X₃ 592 Fc-2382E04 X1 is selectedfrom hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWDAPAGGG YGYYRITYGETGGNSPVQEFTVPVSKGTATISGLKPGVDYTITVYAVEFTFPGAGYYH RPISINYRT-X₃ 593 Fc-2382E05 X1 is selectedfrom hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWDAPAEGG YGYYRITYGETGGNSPVQEFTVPVSKGTATISGLKPGVDYTITVYAVEFDFPGAGYYH RPISINYRT-X₃ 594 Fc-2382E09 X1 is selectedfrom hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWDAPAEAY GYYRITYGETGGNSPVQEFTVPVSKGTATISGLKPGVDYTITVYAVEYDFPGSGYYHR PISINYRT-X₃ 595 Fc-2381A04 X1 is selectedfrom hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWEPFSRLP GGGEYYRITYGETGGNSPLQQFTVPGSKGTATISGLKPGVDYTITVYAVEYPYPFSGY YHRPISINYRT-X₃ 596 Fc-2381A08 X1 isselected from hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWDAPADGG YGYYRITYGETGGNSPVQEFTVPGSKGTATISGLKPGVDYTITVYAVEYDFPGAGYYH RPISINYRT-X₃ 597 Fc-2381B10 X1 is selectedfrom hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWDAPAGGG YGYYRITYGETGGNSPVQEFTVPVSKGTATISGLKPGVDYTITVYAVEYNFIGAGYYH RPISINYRT-X₃ 598 Fc-2381C08 X1 is selectedfrom hinge X₁- Fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWDAPADGA YGYYRITYGETGGNSPVQEFTVPVSKGTATISGLKPGVDYTITVYAVEFPYPFAGYYH RPISINYRT-X₃ 599 Fc-2381G06 X1 is selectedfrom hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWSEKLDGK ARRGYYRITYGETGGNSPVQQFTVPGSKGTATISGLKPGVDYTITVYAVEFPYDHSGY YHRPISINYRT-X₃ 600 Fc-2381H01 X1 isselected from hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWSPRDSTG LVRRGYYRITYGETGGNSPVQQFTVPGSKGTATISGLKPGVDYTITVYAVEYPYDHSG YYHRPISINYRT-X₃ 601 Fc-2381H06 X1 isselected from hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWGDVRTNE ARQGYYRITYGETGGNSPLQGFTVPGSKGTATISGLKPGVDYTITVYAVEYTYEHSGY YHRPISINYRT-X₃ 602 Fc-2381H09 X1 isselected from hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWDAPAGGG YGYYRITYGETGGNSPVQEFTVPVSKGTATISGLKPGVDYTITVYAVEFDFVGAGYYH RPISINYRT-X₃ 603 Fc-2382B11 X1 is selectedfrom hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWDAPAAAY GYYRITYGETGGNSPVQEFTVPVSKGTATISGLKPGVDYTITVYAVEYDFAGSGYYHR PISINYRT-X₃ 604 Fc-2382B08 X1 is selectedfrom hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWDAPADAY GYYRITYGETGGNSPVQEFTVPSSKGTATISGLKPGVDYTITVYAVEFAFPGAGYYHR PISINYRT-X₃ 605 Fc-2382C11 X1 is selectedfrom hinge X₁- Fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWDAPAGGY GYYRITYGETGGNSPVQEFTVPVSKGTATISGLKPGVDYTITVYAVEYDFAGSGYYHR PISINYRT-X₃ 606 Fc-2382G03 X1 is selectedfrom hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWDAPAEAE AYGYYRITYGETGGNSPVQEFTVPVSKGTATISGLKPGVDYTITVYAVEYVFPGAGYY HRPISINYRT-X₃ 607 Fc-2382H03 X1 isselected from hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWDAPAEGA YGYYRITYGETGGNSPVQEFTVPVSKGTATISGLKPGVDYTITVYAVEYPYPFAGYYH RPISINYRT-X₃ 608 Fc-2451A10 X1 is selectedfrom hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VTDVPRDMEVVAATPTSLLISWQPPAVTY GYYRITYGETGGNSTLQQFTVPVYKGTATISGLKPGVDYTITVYAVEYPYDHSGYYHR PISINYRT-X₃ 609 Fc-2451B02 X1 is selectedfrom hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWDAPAAAY GYYRITYGETGGNSPVQEFTVPVSKGTATISGLKPGVDYTITVYAVEFDYPGSGYYHR PISINYRT-X₃ 610 Fc-2451C11 X1 is selectedfrom hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) IVDVPRDLEVVAATPTSLLISWDPPAGAY GYYRITYGETGGNSPKQQFTVPGYKGTATISGLKPGVDYTITVYAVEYPYDHSGYYHR PISINYRT-X₃ 611 Fc-2451H01 X1 is selectedfrom hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWDAPAAGY GYYRITYGETGGNSPVQEFTVPVSKGTATISGLKPGVDYTITVYAVEYDFPGSGYYHR PISINYRT-X₃ 612 Fc-2011B11 X1 is selectedfrom hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWAPPSDAY GYYRITYGETGGNSPVQEFTVPIGKGTATISGLKPGVDYTITVYAVEYPYSHAGYYHR PISINYRT-X₃ 613 Fc-2971A03 X1 is selectedfrom hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWDPPSDDY GYYRITYGETGGNSPVQEFTVPIGKGTATISGLKPGVDYTITVYAVEFPWPHAGYYHR PISINYRT-X₃ 614 Fc-2971A09 X1 is selectedfrom hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWDAPADDY GYYRITYGETGGNSPVQEFTVPIGKGTATISGLKPGVDYTITVYAVEFPWPHAGYYHR PISINYRT-X₃ 615 Fc-2971E02 X1 is selectedfrom hinge X₁- fusion sequences SEQ ID NOs: 317-321;VFLFPPKPKDTLMISRTPEVTCVVVDVSH X2 is selected fromEDPEVKFNWYVDGVEVHNAKTKPREEQYN linker sequences SEQ IDSTYRVVSVLTVLHQDWLNGKEYKCKVSNK NOs: 310-314 and 620-634;ALPAPIEKTISKAKGQPREPQVYTLPPSR X3 is optional and whenDELTKNQVSLTCLVKGFYPSDIAVEWESN present can be a C-terminalGQPENNYKTTPPVLDSDGSFFLYSKLTVD tail sequence selected fromKSRWQQGNVFSCSVMHEALHNHYTQKSLS SEQ ID NOs: 380-395 and LSPG-X₂- EIEK (SEQID NO: 635) VSDVPRDLEVVAATPTSLLISWDAPSDDY GYYRITYGETGGNSPVQEFTVPIGKGTATISGLKPGVDYTITVYAVEFPWPHAGYYHR PISINYRT-X₃

In some embodiments, the anti-PCSK9 Adnectin comprises an Fn3 domain anda PK moiety. In some embodiments, the Fn3 domain is a ¹⁰Fn3 domain. Insome embodiments, the PK moiety increases the serum half-life of thepolypeptide by more than 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,120, 150, 200, 400, 600, 800, 1000% or more relative to the Fn3 domainalone.

In some embodiments, the PK moiety is a polymeric sugar. In someembodiments, the PK moiety is a polyethylene glycol moiety. In someembodiments the PK moiety is a serum albumin binding protein. In someembodiments the PK moiety is human serum albumin. In some embodimentsthe PK moiety is a serum immunoglobulin binding protein. In someembodiments, the PK moiety is transferrin.

In some embodiments the PK moiety is another Adnectin specific for aserum protein, e.g., HSA. The present application provides specificserum albumin binding Adnectin molecules (or SABA), as described herein.In certain embodiments, a PCSK9 Adnectin fused to a SABA can be definedgenerally as follows: X₁-PCSK9 Adnectin core-X₂-X₃-X₄-SABA core-X₅ (SEQID NO: 618), or X₁-SABA core-X₂-X₃-X₄-PCSK9 Adnectin core-X₅ (SEQ ID NO:619), wherein X₁ and X₄ represent optional N-terminal extensionsequences, X₂ and X₅ represent optional C-terminal extension sequences,and X₃ is a linker. In one embodiment, the Adnectins (either PCSK9 orserum albumin binding) of SEQ ID NOs: 618 and 619 comprise the “core”region of Adnectin, i.e., a PCSK9 Adnectin core sequence may be any oneof the PCSK9 Adnectin sequences shown in Table 4, wherein the sequencebegins at the amino acid residue corresponding to E8 of SEQ ID NO: 1 andends at the amino acid corresponding to residue T94 of SEQ ID NO: 1; anda SABA core sequence may be selected from any of the SABA core sequencesshown in Table 6.

In some embodiments, X₁ and X₄ are independently optional, and whenpresent are independently selected from SEQ ID NOs: 371-379 listed inTable 6, and may optionally comprise an M, G or MG sequence at theN-terminus when such residues are not already present. For expression ina mammalian system, the fusion proteins may further comprise a leadersequence at the N-terminus, such as METDTLLLWVLLLWVPGSTG (SEQ ID NO:326). In some embodiments, X₂ and X₅ are independently optional, andwhen present are independently selected from SEQ ID NOs: 380-395 listedin Table 6. In certain embodiments, X₃ is a linker sequence selectedfrom SEQ ID NOs: 396-419 listed in Table 6. The sequences shown in Table2 represent exemplary fusions of anti-PCSK9 Adnectin and SABA. It shouldbe understood that any PCSK9 Adnectin and SABA sequence described in thepresent application may be incorporated into these configurations.

TABLE 2 Exemplary PCSK9 Adnectin - SABA Fusion Sequences SEQ ID Clone orName Description Sequence 616 PCSK9 PCSK9 Adnectin sequence is X₁-Adnectin- underlined; SABA sequence is EVVAATPTSLLISWVPPSDDYGYYRITY SABAfusion in bold. PCSK9 Adnectin GETGGNSPVQEFTVPIGKGTATISGLKP sequence isthe core region GVDYTITVYAVEFPWPHAGYYHRPISIN derived from clone 2013E01;YRT-X₂-X₃-X₄- SABA sequence is derived EVVAATPTSLLISWHSYYEQNSYYRITY fromSABA 1 (SEQ ID NO: GETGGNSPVQEFTVPYSQTTATISGLKP 330)GVDYTITVYAVYGSKYYYPISINYRT- X₅ 617 SABA-PCSK9 SABA sequence is in bold;X₁- Adnectin PCSK9 Adnectin sequence is EVVAATPTSLLISWHSYYEQNSYYRITYfusion underlined. SABA sequence GETGGNSPVQEFTVPYSQTTATISGLKP is derivedfrom SABA 1 (SEQ GVDYTITVYAVYGSKYYYPISINYRT- ID NO: 330); PCSK9X₂-X₃-X₄- Adnectin sequence is the core EVVAATPTSLLISWVPPSDDYGYYRITYregion derived from clone GETGGNSPVQEFTVPIGKGTATISGLKP 2013E01GVDYTITVYAVEFPWPHAGYYHRPISIN YRT-X₅

The application provides Adnectin comprising a Fn3 domain that binds toPCSK9. Polypeptide binding to a target molecule may be assessed in termsof equilibrium constants (e.g., dissociation, K_(D)) and in terms ofkinetic constants (e.g., on-rate constant, K_(on) and off-rate constant,k_(off)). An Adnectin will generally bind to a target molecule with aK_(D) of less than 500 nM, 100 nM, 10 nM, 1 nM, 500 pM, 200 pM, 100 pM,although higher K_(D) values may be tolerated where the K_(off) issufficiently low or the K_(on), is sufficiently high.

The SEQ ID NOS of the BC, DE and FG loops of the anti-PCSK9 Adnectins ofthe invention are presented in italic in Table 3.

TABLE 3 Anti-PCSK9 Adnectin BC, DE and FG Loops LDLR PCSK9- DepletionEGFA FRET (% inhibition at Affinity (EC₅₀, 75 nM, EC₅₀ SEQ ID SEQ ID SEQID Clone ID (K_(D), nM) nM) (nM)) BC Loop NO DE Loop NO FG loop NO1459D05 14.4† 4  66.8, >200 SWPPPSHGYG 2 PPGKGT 18 EYPYKHSGYYHRP 28 1.58* 26{circumflex over ( )} 1784F03  3.8{circumflex over ( )} 2150.2, 26 ± 13 SWRPPIHAYG 3 PIVEGT 19 EYTFKHSGYYHRP 29 1784F03-m1 nd ndnd, >2000 SWDAPIHAYG 4 PGSEGT 20 EYTFKHSGYYHRP 29 1784F03-m2 nd ndnd, >2000 SWDAPAHAYG 5 PGSKGT 21 EYTFKHSGYYHRP 29 1784F03-m3 nd ndnd, >2000 SWDAPAVTYG 6 PGSKST 22 EYTFKHSGYYHRP 29 1813E02 <2{circumflexover ( )} 1.3 nd, 16 SWSPPANGYG 7 PVGRGT 23 EYTYKGSGYYHRP 30 1923B02 0.173* 2.3 178.0, 23 ± 7 SWTPPPKGYG 8 PVGEGT 24 EYTYNGAGYYHRP 311923B02(N82I) nd nd nd, 14 SWTPPPKGYG 8 PVGEGT 24 EYTYIGAGYYHRP 321923B02(N82E) nd nd nd, 28 SWTPPPKGYG 8 PVGEGT 24 EYTYEGAGYYHRP 331923B02(T80A) nd nd nd, 42 SWTPPPKGYG 8 PVGEGT 24 EYAYNGAGYYHRP 341922G04  0.09* 1.2 105.1, 10 ± 2 SWRPPSHAYG 9 PIGKGT 25 EYPWKGSGYYHRP 351922G04(R25D) nd 2.5 nd, 29 ± 8 SWDPPSHAYG 10 PIGKGT 25 EYPWKGSGYYHRP 351922G04(R25E) nd 3.5 nd, 29 ± 18 SWEPPSHAYG 11 PIGKGT 25 EYPWKGSGYYHRP35 1922G04(R25S) nd nd nd, 21 SWSPPSHAYG 12 PIGKGT 25 EYPWKGSGYYHRP 352012A04  0.25* 2.1 144.5, 12 ± 6 SWRPPSNGHG 13 PVNEGT 26 EFPFKWSGYYHRP36 2013E01  1.51† 1.6 165.5, 10 ± 4 SWVPPSDDYG 14 PIGKGT 25EFPWPHAGYYHRP 37  0.29* 2011H05  0.08* 2.7 197.6, 12 ± 5 SWVPSSHAYG 15PVGVGT 27 EYAFEGAGYYHRP 38 2011H05(V23D) nd 5.5 nd, 18 ± 3 SWDPSSHAYG 16PVGVGT 27 EYAFEGAGYYHRP 38 2011H05(V23E) nd 7.4 nd, 12 ± 3 SWEPSSHAYG 17PVGVGT 27 EYAFEGAGYYHRP 38 2381B02(1)  3.29† 2.5 125.4, nd SWEPFSRLPGGGE106 PGSKGT 21 EYPYDYSGYYHRP 142 2381B04(1)  0.527† 2.4 121.6, ndSWEPFSRLPGGGE 106 PGSKGT 21 EYPYEHSGYYHRP 143 2381B06(1) nd 3.5 119.7,nd SWEPFSRLPGGGE 106 PGSKGT 21 EYPYPHSGYYHRP 144 2381B08  4.11† 2.6124.8, nd SWDAPADGGYG 107 PSSKGT 136 EYTFPGAGYYHRP 145 2381D02(1) nd 3.1185.0, nd SWEPFSRLPGGGE 106 PGSKGT 21 EYPYDHSGYYHRP 146 2381D04(1) 0.237† 2.9 119.2, nd SWEPFSRLPGGGE 106 PGSKGT 21 EFPYDHSGYYHRP 1472381F11  1.59† 4 110.2, nd SWDAPADGGYG 107 PVSKST 137 EYTFPGAGYYHRP 1452381G03(1) nd 3.4  70.2, nd SWEPFSRLPGGGE 106 PGSKGT 21 EFPYAHSGYYHRP148 2381G09  1.12† 3.1 133.0, nd SWDAPAGDGYG 108 PVSKGT 138EFTFPGAGYYHRP 149 2381H03(1) nd 3.4  89.8, nd SWEPFSRLPGGGE 106 PGSKGT21 EYPYAHSGYFHRP 150 2382A01 nd 12.9 119.8, nd SWAAPAGGGYG 109 PVSKGT138 EYDFPGAGYYHRP 151 2382B10  2.35† 3 100.2, nd SWDAPADAYG 110 PSSKGT136 EYDFPGSGYYHRP 152 2382B09  0.656† 3.8 105.0, nd SWDAPADAYG 110PVSKGT 138 EFDYPGSGYYHRP 153 2382C05  2.49† 4 105.3, nd SWDAPADGAYG 111PVSKGT 138 EYSFPGAGYYHRP 154 2382C09  0.757† 3.5 121.7, nd SWDAPAEGYG112 PVSKGT 138 EFDFPGSGYYHRP 155 2382D03  1.53† 3.3  80.4, ndSWDAPADEAYG 113 PVSKGT 138 EFDFPGAGYYHRP 156 2382D05  0.314† 2.6 140.5,nd SWDAPADGGYG 107 PVSKGT 138 EFDFPGAGYYHRP 156 2382D08 nd 3.1 106.6, ndSWDAPADGYG 114 PVSKGT 138 EFPFPGSGYYHRP 157 2382D09  0.304† 2.6 109.1,nd SWDAPAEGYG 112 PVSKGT 138 EFDFPGAGYYHRP 156 2382F02 nd 2.6  −6.3, ndSWDAPAGGGYG 115 PVSKGT 138 EFDFPGSGYYHRP 155 2382F03 nd 2.7  88.6, ndSWDAPAADAYG 116 PVSKGT 138 EFNFPGAGYYHRP 158 2382F05  4.54† 2.4  72.2,nd SWDAPAEAGKHYG 117 PVSKGT 138 EFDFPGAGYYHRP 156 2382F08 nd 2.5 105.0,nd SWDAPAEAYG 118 PVSKGT 138 EFTYPGSGYYHRP 159 2382F09 nd 3.1 109.7, ndSWDAPAAAYG 119 PVSKGT 138 EYDFPGSGYYHRP 152 2382G04  1.11† 2.9 146.1, ndSWDAPAGGGYG 115 PSSKGT 136 EFDFPGAGYYHRP 156 2382H10  1.40† 2.6 118.6,nd SWDAPAGGYG 120 PVSKGT 138 EFDFPGSGYYHRP 155 2382H11 nd 2.9 117.2, ndSWDAPADGYG 114 PVFKGT 139 EFDYPGSGYYHRP 153 2382H04 nd 3.2  68.2, ndSWDAPAAGGYG 121 PSSKGT 136 EYDFPGAGYYHRP 151 2382H07 nd 2.7  86.2, ndSWDAPADAYG 110 PGSKGT 21 EFDFPGSGYYHRP 155 2382H09  1.86† 0.9 101.2, ndSWDAPAAAYG 119 PSSKGT 136 EFDFPGSGYYHRP 155 2451A02 nd 3.2 106.4, ndSWDAPAAGYG 122 PVSKGT 138 EFPFPGSGYYHRP 157 2451B05 nd 6.3  91.7, ndSWDAPAGGYG 120 PSSKGT 136 EFDYPGSGYYHRP 153 2451B06 nd 4.5  92.2, ndSWDAPADGGYG 107 PVSKGT 138 EFDFPGAGYYHRP 156 2451C06  1.27† 1.2  89.4,nd SWDAPAGAASYG 123 PVSKGT 138 EFPFPGAGYYHRP 160 2451D05 nd 2.8 115.0,nd SWDAPAGAYG 124 PVSKGT 138 EFDFPGSGYYHRP 155 2451F03 nd 2.8 113.2, ndSWDPPAEGYG 125 PVSKGT 138 EFNFPGSGYYHRP 161 2451G01 nd 3.8  90.8, ndSWDAPAGGYG 120 PSSKGT 136 EFDFPGSGYYHRP 155 2451H07(2)  2.08† 0.2  88.8,nd SWNPPDVNYG 126 PVSKGT 138 EYPYAHAGYYHRP 162 2382E03  2.94† 2.4  89.5,nd SWDAPAGDGYG 108 PVSKGT 138 EFDFPGAGYYHRP 156 2382E04 nd 3  61.5, ndSWDAPAGGGYG 115 PVSKGT 138 EFTFPGAGYYHRP 149 2382E05  0.604† 2.8 103.5,nd SWDAPAEGGYG 127 PVSKGT 138 EFDFPGAGYYHRP 156 2382E09 nd 6.2  97.2, ndSWDAPAEAYG 118 PVSKGT 138 EYDFPGSGYYHRP 152 2381A04(1) nd 3.3 100.1, ndSWEPFSRLPGGGE 106 PGSKGT 21 EYPYPFSGYYHRP 163 2381A08 nd 3.6  91.4, ndSWDAPADGGYG 107 PGSKGT 21 EYDFPGAGYYHRP 151 2381B10 nd 7.3  96.4, ndSWDAPAGGGYG 115 PVSKGT 138 EYNFIGAGYYHRP 164 2381C08 nd 0.7  15.3, ndSWDAPADGAYG 111 PVSKGT 138 EFPYPFAGYYHRP 165 2381G06(3) nd 9  57.7, ndSWSEKLDGKARRG 128 PGSKGT 21 EFPYDHSGYYHRP 147 2381H01(3) nd 4  22.2, ndSWSPRDSTGLVRRG 129 PGSKGT 21 EYPYDHSGYYHRP 146 2381H06(4) nd 5  53.4, ndSWGDVRTNEARQG 130 PGSKGT 21 EYTYEHSGYYHRP 166 2381H09  3.23† 3.4  94.4,nd SWDAPAGGGYG 115 PVSKGT 138 EFDFVGAGYYHRP 167 2382B11 nd 2.9  88.8, ndSWDAPAAAYG 119 PVSKGT 138 EYDFAGSGYYHRP 168 2382B08 nd 2.9 107.2, ndSWDAPADAYG 110 PSSKGT 136 EFAFPGAGYYHRP 169 2382C11 nd 3.7  82.9, ndSWDAPAGGYG 120 PVSKGT 138 EYDFAGSGYYHRP 168 2382G03 nd 2.7  77.8, ndSWDAPAEAEAYG 131 PVSKGT 138 EYVFPGAGYYHRP 170 2382H03  0.677† 3.4 102.1,nd SWDAPAEGAYG 132 PVSKGT 138 EYPYPFAGYYHRP 171 2451A10(5) nd 10.9 53.7, nd SWQPPAVTYG 133 PVYKGT 140 EYPYDHSGYYHRP 146 2451B02 nd 5.3 71.4, nd SWDAPAAAYG 119 PVSKGT 138 EFDYPGSGYYHRP 153 2451C11(6) nd 9.7 70.3, nd SWDPPAGAYG 134 PGYKGT 141 EYPYDHSGYYHRP 146 2451H01 nd 2.8 95.8, nd SWDAPAAGYG 122 PVSKGT 138 EYDFPGSGYYHRP 152 2011B11 nd 1.7144.5, nd SWAPPSDAYG 135 PIGKGT 25 EYPYSHAGYYHRP 172 2971A03  0.806† nd120.1, nd SWDPPSDDYG 301 PIGKGT 25 EFPWPHAGYYHRP 37 2971A09  2.79† nd132.3, nd SWDAPADDYG 302 PIGKGT 25 EFPWPHAGYYHRP 37 2971E02  1.78† nd126.2, nd SWDAPSDDYG 303 PIGKGT 25 EFPWPHAGYYHRP 37 †K_(D) determinedusing Octet Red at 37° C.; *K_(D) determined using ProteOn at 25° C.;{circumflex over ( )}K_(D) determined using ITC at 37° C.; (1)Inaddition to mutations in the loops, these clones also have the mutationsV45L and E47Q; (2)In addition to mutations in the loops, this clone alsohas the mutations V1I, S2T, and V45L; (3)In addition to mutations in theloops, these clones also have the mutation E47Q; (4)In addition tomutations in the loops, this clone also has the mutations V45L and E47G;(5)In addition to mutations in the loops, this clone also has themutations S2T, L8M, P44T, V45L, and E47Q; (6)In addition to mutations inthe loops, this clone also has the mutations VII, S2V, V45K, and E47Q.

The SEQ ID NOS of the family of anti-PCSK9 Adnectin of the invention arepresented in Table 4.

TABLE 4 Anti-PCSK9 Adnectin Family Sequence Clone Amino Acid NucleicAcid 1459D05 also MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGAreferred to as SWPPPSHGYGYYRITYGETGGNTCAGCTGGCCGCCGCCGTCTCATGGTTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGATI000891 or SPVQEFTVPPGKGTATISGLKPCAATAGCCCTGTCCAGGAGTTCACTGTGCCGCCTGGTAAAGGTACAGCTACCATCAGCGGCCTT ATI-891GVDYTITVYAVEYPYKHSGYYHAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATACCCGTACAAACATTCTGGTTRPISINYRTEIDKPSQHHHHHHACTACCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCA (SEQ IDNO: 39) CCACCACCAC (SEQ ID NO: 40) 1784F03 MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWRPPIHAYGYYRITYGETGGNTCAGCTGGAGGCCGCCGATTCATGCTTACGGGTATTACCGCATCACTTACGGCGAAACAGGAGGSPVQEFTVPIVEGTATISGLKPCAATAGCCCTGTCCAGGAGTTCACTGTGCCTATTGTTGAAGGTACAGCTACCATCAGCGGCCTTGVDYTITVYAVEYTFKHSGYYHAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATATACATTTAAACATTCCGGTTRPISINYRTEIDKPSQHHHHHHACTACCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCA (SEQ IDNO: 41) CCACCACCAC (SEQ ID NO: 42) 1784F03-m1 MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWDAPIHAYGYYRITYGETGGNTCAGCTGGGACGCTCCGATTCATGCTTACGGGTATTACCGCATCACTTACGGCGAAACAGGAGGSPVQEFTVPGSEGTATISGLKPCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGGTTCTGAAGGTACAGCTACCATCAGCGGCCTTGVDYTITVYAVEYTFKHSGYYHAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATATACATTTAAACATTCCGGTTRPISINYRTEIDKPSQHHHHHHACTACCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCA (SEQ IDNO: 43) CCACCACCAC (SEQ ID NO: 44) 1784F03-m2 MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWDAPAHAYGYYRITYGETGGNTCAGCTGGGACGCTCCGGCTCATGCTTACGGGTATTACCGCATCACTTACGGCGAAACAGGAGGSPVQEFTVPGSKGTATISGLKPCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGGTTCTAAAGGTACAGCTACCATCAGCGGCCTTGVDYTITVYAVEYTFKHSGYYHAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATATACATTTAAACATTCCGGTTRPISINYRTEIDKPSQHHHHHHACTACCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCA (SEQ IDNO: 45) CCACCACCAC (SEQ ID NO: 46) 1784F03-m3 MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWDAPAVTYGYYRITYGETGGNTCAGCTGGGACGCTCCGGCTGTTACTTACGGGTATTACCGCATCACTTACGGCGAAACAGGAGGSPVQEFTVPGSKSTATISGLKPCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGGTTCTAAATCTACAGCTACCATCAGCGGCCTTGVDYTITVYAVEYTFKHSGYYHAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATATACATTTAAACATTCCGGTTRPISINYRTEIDKPSQHHHHHHACTACCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCA (SEQ IDNO: 47) CCACCACCAC (SEQ ID NO: 48) 1813E02 MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWSPPANGYGYYRITYGETGGNTCAGCTGGTCCCCACCGGCTAACGGTTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGSPVQEFTVPVGRGTATISGLKPCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTGGTAGAGGTACAGCTACCATCAGCGGCCTTGVDYTITVYAVEYTYKGSGYYHAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAGTATACCTACAAAGGCTCTGGTTRPISINYRTEIDKPSQHHHHHHACTACCATCGCCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCA (SEQ IDNO: 49) CCACCACCAC (SEQ ID NO: 50) 1923B02 MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWTPPPKGYGYYRITYGETGGNTCAGCTGGACGCCTCCCCCTAAAGGGTATGGTTATTACCGCATCACTTACGGCGAAACAGGAGGSPVQEFTVPVGEGTATISGLKPCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTGGTGAAGGTACAGCTACCATCAGCGGCCTTGVDYTITVYAVEYTYNGAGYYHAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATACACGTACAACGGTGCCGGTTRPISINYRTEIDKPSQHHHHHHACTACCACCGGCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCA (SEQ IDNO: 51) CCACCACCAC (SEQ ID NO: 52) 1923B02(N82I) MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWTPPPKGYGYYRITYGETGGNTCAGCTGGACGCCTCCCCCTAAAGGGTATGGTTATTACCGCATCACTTACGGCGAAACAGGAGGSPVQEFTVPVGEGTATISGLKPCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTGGTGAAGGTACAGCTACCATCAGCGGCCTTGVDYTITVYAVEYTYIGAGYYHAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATACACGTACATTGGTGCCGGTTRPISINYRTGSGSHHHHHHACTACCACCGGCCAATTTCCATTAATTACCGCACAGGTAGCGGTTCCCACCATCACCACCATCA (SEQ IDNO: 53) C (SEQ ID NO: 54) 1923B02(N82E) MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWTPPPKGYGYYRITYGETGGNTCAGCTGGACGCCTCCCCCTAAAGGGTATGGTTATTACCGCATCACTTACGGCGAAACAGGAGGSPVQEFTVPVGEGTATISGLKPCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTGGTGAAGGTACAGCTACCATCAGCGGCCTTGVDYTITVYAVEYTYEGAGYYHAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATACACGTACGAAGGTGCCGGTTRPISINYRTGSGSHHHHHHACTACCACCGGCCAATTTCCATTAATTACCGCACAGGTAGCGGTTCCCACCATCACCACCATCA (SEQ IDNO: 55) C (SEQ ID NO: 56) 1923B02(T80A) MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWTPPPKGYGYYRITYGETGGNTCAGCTGGACGCCTCCCCCTAAAGGGTATGGTTATTACCGCATCACTTACGGCGAAACAGGAGGSPVQEFTVPVGEGTATISGLKPCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTGGTGAAGGTACAGCTACCATCAGCGGCCTTGVDYTITVYAVEYAYNGAGYYHAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATACGCGTACAACGGTGCCGGTTRPISINYRTGSGSHHHHHHACTACCACCGGCCAATTTCCATTAATTACCGCACAGGTAGCGGTTCCCACCATCACCACCATCA (SEQ IDNO: 57) C (SEQ ID NO: 58) 1922G04 also MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGAreferred to SWRPPSHAYGYYRITYGETGGNTCAGCTGGCGGCCGCCATCTCATGCTTATGGTTATTACCGCATCACTTACGGCGAAACAGGAGG hereinSPVQEFTVPIGKGTATISGLKPCAATAGCCCTGTCCAGGAGTTCACTGTGCCTATTGGGAAAGGTACAGCTACCATCAGCGGCCTT asATI001057 GVDYTITVYAVEYPWKGSGYYHAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATACCCGTGGAAAGGTTCTGGTT orATI-1057 RPISINYRTEIDKPSQHHHHHHACTACCATCGGCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCA (SEQ IDNO: 59) CCACCACCAC (SEQ ID NO: 60) 1922G04(R25D) MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWDPPSHAYGYYRITYGETGGNTCAGCTGGGACCCGCCATCTCATGCTTATGGTTATTACCGCATCACTTACGGCGAAACAGGAGGSPVQEFTVPIGKGTATISGLKPCAATAGCCCTGTCCAGGAGTTCACTGTGCCTATTGGGAAAGGTACAGCTACCATCAGCGGCCTTGVDYTITVYAVEYPWKGSGYYHAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATACCCGTGGAAAGGTTCTGGTTRPISINYRTGSGSHHHHHHACTACCATCGGCCAATTTCCATTAATTACCGCACAGGTAGCGGTTCCCACCATCACCACCATCA (SEQ IDNO: 61) C (SEQ ID NO: 62) 1922G04(R25E) MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWEPPSHAYGYYRITYGETGGNTCAGCTGGGAACCGCCATCTCATGCTTATGGTTATTACCGCATCACTTACGGCGAAACAGGAGGSPVQEFTVPIGKGTATISGLKPCAATAGCCCTGTCCAGGAGTTCACTGTGCCTATTGGGAAAGGTACAGCTACCATCAGCGGCCTTGVDYTITVYAVEYPWKGSGYYHAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATACCCGTGGAAAGGTTCTGGTTRPISINYRTGSGSHHHHHHACTACCATCGGCCAATTTCCATTAATTACCGCACAGGTAGCGGTTCCCACCATCACCACCATCA (SEQ IDNO: 63) C (SEQ ID NO: 64) 1922G04(R25S) MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWSPPSHAYGYYRITYGETGGNTCAGCTGGAGCCCGCCATCTCATGCTTATGGTTATTACCGCATCACTTACGGCGAAACAGGAGGSPVQEFTVPIGKGTATISGLKPCAATAGCCCTGTCCAGGAGTTCACTGTGCCTATTGGGAAAGGTACAGCTACCATCAGCGGCCTTGVDYTITVYAVEYPWKGSGYYHAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATACCCGTGGAAAGGTTCTGGTTRPISINYRTGSGSHHHHHHACTACCATCGGCCAATTTCCATTAATTACCGCACAGGTAGCGGTTCCCACCATCACCACCATCA (SEQ IDNO: 65) C (SEQ ID NO: 66) 2012A04 MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWRPPSNGHGYYRITYGETGGNTCAGCTGGCGGCCCCCCTCTAATGGTCACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGSPVQEFTVPVNEGTATISGLKPCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTAATGAAGGTACAGCTACCATCAGCGGCCTTGVDYTITVYAVEFPFKWSGYYHAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATTCCCCTTCAAGTGGTCGGGCTRPISINYRTEIDKPSQHHHHHHACTACCATCGACCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCA (SEQ IDNO: 67) CCACCACCAC (SEQ ID NO: 68) 2013E01 also MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGAreferred to as SWVPPSDDYGYYRITYGETGGNTCAGCTGGGTCCCGCCTTCAGATGATTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGATI001081 or SPVQEFTVPIGKGTATISGLKPCAATAGCCCTGTCCAGGAGTTCACTGTGCCTATTGGTAAAGGAACAGCTACCATCAGCGGCCTTATI-1081 GVDYTITVYAVEFPWPHAGYYHAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAGTTTCCGTGGCCACATGCTGGTTRPISINYRTEIDKPSQHHHHHHACTATCATCGGCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCA (SEQ IDNO: 69) CCACCACCAC (SEQ ID NO: 70) 2011H05 also MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGAreferred to as SWVPSSHAYGYYRITYGETGGNTCAGCTGGGTTCCGTCGTCTCATGCCTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGATI001091 or SPVQEFTVPVGVGTATISGLKPCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTGGGGGTAGGTACAGCTACCATCAGCGGCCTTATI-1091 GVDYTITVYAVEYAFEGAGYYHAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATACGCTTTCGAAGGGGCTGGTTRPISINYRTEIDKPSQHHHHHHACTACCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCA (SEQ IDNO: 71) CCACCACCAC (SEQ ID NO: 72) 2011H05(V23D) MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWDPSSHAYGYYRITYGETGGNTCAGCTGGGACCCGTCGTCTCATGCCTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGSPVQEFTVPVGVGTATISGLKPCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTGGGGGTAGGTACAGCTACCATCAGCGGCCTTGVDYTITVYAVEYAFEGAGYYHAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATACGCTTTCGAAGGGGCTGGTTRPISINYRTEIDKPSQHHHHHHACTACCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCA (SEQ IDNO: 73) CCACCACCAC (SEQ ID NO: 74) 2011H05(V23E) MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWEPSSHAYGYYRITYGETGGNTCAGCTGGGAACCGTCGTCTCATGCCTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGSPVQEFTVPVGVGTATISGLKPCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTGGGGGTAGGTACAGCTACCATCAGCGGCCTTGVDYTITVYAVEYAFEGAGYYHAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATACGCTTTCGAAGGGGCTGGTTRPISINYRTEIDKPSQHHHHHHACTACCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCA (SEQ IDNO: 75) CCACCACCAC (SEQ ID NO: 76) 2381B02 MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWEPFSRLPGGGEYYRITYGETTCAGCTGGGAGCCGTTCAGCCGGTTGCCCGGGGGCGGCGAGTATTACCGGATCACTTACGGCGAGGNSPLQQFTVPGSKGTATISGAACAGGAGGCAATAGCCCTCTGCAGCAGTTCACTGTGCCTGGTTCTAAAGGTACAGCTACCATCLKPGVDYTITVYAVEYPYDYSGAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATACCCGTACGACTYYHRPISINYRTEIDKPSQHHHATTCTGGTTACTACCATCGCCCCATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCA HHH(SEQ ID NO: 173) GCACCATCACCACCACCAC (SEQ ID NO: 174) 2381B04MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWEPFSRLPGGGEYYRITYGETTCAGCTGGGAGCCGTTCAGCCGGTTGCCCGGGGGCGGCGAGTATTACCGGATCACTTACGGCGAGGNSPLQQFTVPGSKGTATISGAACAGGAGGCAATAGCCCTCTGCAGCAGTTCACTGTGCCTGGTTCTAAAGGTACAGCTACCATCLKPGVDYTITVYAVEYPYEHSGAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATACCCGTACGAGCYYHRPISINYRTEIDKPSQHHHATTCTGGGTACTATCATCGTCCGATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCA HHH(SEQ ID NO: 175) GCACCATCACCACCACCAC (SEQ ID NO: 176) 2381B06MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWEPFSRLPGGGEYYRITYGETTCAGCTGGGAGCCGTTCAGCCGGTTGCCCGGGGGCGGCGAGTATTACCGGATCACTTACGGCGAGGNSPLQQFTVPGSKGTATISGAACAGGAGGCAATAGCCCTCTGCAGCAGTTCACTGTGCCTGGTTCTAAAGGTACAGCTACCATCLKPGVDYTITVYAVEYPYPHSGAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATACCCGTACCCGCYYHRPISINYRTEIDKPSQHHHATTCTGGTTACTACCATCGACCGATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCA HHH(SEQ ID NO: 177) GCACCATCACCACCACCAC (SEQ ID NO: 178) 2381B08MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWDAPADGGYGYYRITYGETGGTCAGCTGGGACGCTCCGGCTGATGGAGGGTACGGTTATTACCGCATCACTTACGGCGAAACAGGNSPVQEFTVPSSKGTATISGLKAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTAGTTCTAAAGGTACAGCTACCATCAGCGGCPGVDYTITVYAVEYTFPGAGYYCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATACACCTTCCCGGGCGCCGHRPISINYRTEIDKPSQHHHHHGTTACTACCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCA H (SEQID NO: 179) TCACCACCACCAC (SEQ ID NO: 180) 2381D02MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWEPFSRLPGGGEYYRITYGETTCAGCTGGGAGCCGTTCAGCCGGTTGCCCGGGGGCGGCGAGTATTACCGGATCACTTACGGCGAGGNSPLQQFTVPGSKGTATISGAACAGGAGGCAATAGCCCTCTGCAGCAGTTCACTGTGCCTGGTTCTAAAGGTACAGCTACCATCLKPGVDYTITVYAVEYPYDHSGAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATACCCGTACGACCYYHRPISINYRTEIDKPSQHHHATTCTGGTTACTACCATCGTCCCATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCA HHH(SEQ ID NO: 181) GCACCATCACCACCACCAC (SEQ ID NO: 182) 2381D04MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWEPFSRLPGGGEYYRITYGETTCAGCTGGGAGCCGTTCAGCCGGTTGCCCGGGGGCGGCGAGTATTACCGGATCACTTACGGCGAGGNSPLQQFTVPGSKGTATISGAACAGGAGGCAATAGCCCTCTGCAGCAGTTCACTGTGCCTGGTTCTAAAGGTACAGCTACCATCLKPGVDYTITVYAVEFPYDHSGAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATTCCCGTACGACCYYHRPISINYRTEIDKPSQHHHATTCTGGTTACTACCATCGGCCCATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCA HHH(SEQ ID NO: 183) GCACCATCACCACCACCAC (SEQ ID NO: 184) 2381F11MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWDAPADGGYGYYRITYGETGGTCAGCTGGGACGCTCCGGCTGACGGGGGGTACGGTTATTACCGCATCACTTACGGCGAAACAGGNSPVQEFTVPVSKSTATISGLKAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAAGTACAGCTACCATCAGCGGCPGVDYTITVYAVEYTFPGAGYYCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATACACCTTCCCCGGCGCCGHRPISINYRTEIDKPSQHHHHHGTTACTACCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCA H (SEQID NO: 185) TCACCACCACCAC (SEQ ID NO: 186) 2381G03MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWEPFSRLPGGGEYYRITYGETTCAGCTGGGAGCCGTTCAGCCGGTTGCCCGGGGGCGGCGAGTATTACCGGATCACTTACGGCGAGGNSPLQQFTVPGSKGTATISGAACAGGAGGCAATAGCCCTCTGCAGCAGTTCACTGTGCCTGGTTCTAAAGGTACAGCTACCATCLKPGVDYTITVYAVEFPYAHSGAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATTCCCGTACGCGCYYHRPISINYRTEIDKPSQHHHATTCTGGGTACTACCATCGTCCGATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCA HHH(SEQ ID NO: 187) GCACCATCACCACCACCAC (SEQ ID NO: 188) 2381G09MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWDAPAGDGYGYYRITYGETGGTCAGCTGGGACGCTCCGGCTGGGGACGGTTACGGTTATTACCGCATCACTTACGGCGAAACAGGNSPVQEFTVPVSKGTATISGLKAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCCGTTTCTAAAGGTACAGCTACCATCAGCGGCPGVDYTITVYAVEFTFPGAGYYCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATTCACCTTCCCGGGCGCCGHRPISINYRTEIDKPSQHHHHHGTTACTACCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCA H (SEQID NO: 189) TCACCACCACCAC (SEQ ID NO: 190) 2381H03MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWEPFSRLPGGGEYYRITYGETTCAGCTGGGAGCCGTTCAGCCGGTTGCCCGGGGGCGGCGAGTATTACCGGATCACTTACGGCGAGGNSPLQQFTVPGSKGTATISGAACAGGAGGCAATAGCCCTCTGCAGCAGTTCACTGTGCCTGGTTCTAAAGGTACAGCTACCATCLKPGVDYTITVYAVEYPYAHSGAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATACCCGTACGCGCYFHRPISINYRTEIDKPSQHHHATTCTGGTTACTTCCATCGTCCGATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCA HHH(SEQ ID NO: 191) GCACCATCACCACCACCAC (SEQ ID NO: 192) 2382A01MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWAAPAGGGYGYYRITYGETGGTCAGCTGGGCCGCTCCGGCTGGTGGTGGCTACGGTTATTACCGCATCACTTACGGCGAAACAGGNSPVQEFTVPVSKGTATISGLKAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCPGVDYTITVYAVEYDFPGAGYYCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATACGACTTCCCGGGCGCCGHRPISINYRTEIDKPSQHHHHHGTTACTACCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCA H (SEQID NO: 193) TCACCACCACCAC (SEQ ID NO: 194) 2382B10MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWDAPADAYGYYRITYGETGGNTAAGCTGGGACGCTCCGGCTGACGCGTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGSPVQEFTVPSSKGTATISGLKPCAATAGCCCTGTCCAGGAGTTCACTGTGCCTAGTTCTAAAGGTACAGCTACCATCAGCGGCCTTGVDYTITVYAVEYDFPGSGYYHAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATACGACTTCCCCGGCAGCGGTTRPISINYRTEIDKPSQHHHHHHACTACCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCA (SEQ IDNO: 195) CCACCACCAC (SEQ ID NO: 196) 2382B09 MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWDAPADAYGYYRITYGETGGNTCAGCTGGGACGCTCCGGCTGACGCGTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGSPVQEFTVPVSKGTATISGLKPCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCCTTGVDYTITVYAVEFDYPGSGYYHAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATTCGACTACCCCGGCTCCGGTTRPISINYRTEIDKPSQHHHHHHACTACCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCA (SEQ IDNO: 197) CCACCACCAC (SEQ ID NO: 198) 2382C05 MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWDAPADGAYGYYRITYGETGGTCAGCTGGGACGCTCCGGCTGATGGGGCATACGGTTATTACCGCATCACTTACGGCGAAACAGGNSPVQEFTVPVSKGTATISGLKAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAGGGTACAGCTACCATCAGCGGCPGVDYTITVYAVEYSFPGAGYYCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATACTCCTTCCCCGGCGCCGHRPISINYRTEIDKPSQHHHHHGTTACTACCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCA H (SEQID NO: 199) TCACCACCACCAC (SEQ ID NO: 200) 2382C09MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWDAPAEGYGYYRITYGETGGNTCAGCTGGGACGCTCCGGCTGAGGGTTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGSPVQEFTVPVSKGTATISGLKPCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCCTTGVDYTITVYAVEFDFPGSGYYHAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATTCGACTTCCCCGGCTCCGGTTRPISINYRTEIDKPSQHHHHHHACTACCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCA (SEQ IDNO: 201) CCACCACCAC (SEQ ID NO: 202) 2382D03 MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWDAPADEAYGYYRITYGETGGTCAGCTGGGACGCTCCGGCTGACGAGGCGTACGGTTATTACCGCATCACTTACGGCGAAACAGGNSPVQEFTVPVSKGTATISGLKAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCPGVDYTITVYAVEFDFPGAGYYCTTAAACCTGGTGTTGATTATACCATCACTGTGTATGCTGTCGAATTCGACTTCCCCGGCGCCGHRPISINYRTEIDKPSQHHHHHGTTACTACCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCA H (SEQID NO: 203) TCACCACCACCAC (SEQ ID NO: 204) 2382D05MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWDAPADGGYGYYRITYGETGGTCAGCTGGGACGCTCCGGCTGATGGTGGTTACGGTTATTACCGCATCACTTACGGCGAAACAGGNSPVQEFTVPVSKGTATISGLKAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCPGVDYTITVYAVEFDFPGAGYYCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATTCGACTTCCCGGGCGCCGHRPISINYRTEIDKPSQHHHHHGTTACTACCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCA H (SEQID NO: 205) TCACCACCACCAC (SEQ ID NO: 206) 2382D08MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWDAPADGYGYYRITYGETGGNTCAGCTGGGACGCTCCGGCTGATGGCTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGSPVQEFTVPVSKGTATISGLKPCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCCTTGVDYTITVYAVEFPFPGSGYYHAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATTCCCCTTCCCCGGCTCCGGTTRPISINYRTEIDKPSQHHHHHHACTACCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCA (SEQ IDNO: 207) CCACCACCAC (SEQ ID NO: 208) 2382D09 MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWDAPAEGYGYYRITYGETGGNTCAGCTGGGACGCTCCGGCTGAAGGGTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGSPVQEFTVPVSKGTATISGLKPCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCCTTGVDYTITVYAVEFDFPGAGYYHAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATTCGACTTCCCCGGCGCCGGTTRPISINYRTEIDKPSQHHHHHHACTACCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCA (SEQ IDNO: 209) CCACCACCAC (SEQ ID NO: 210) 2382F02 MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWDAPAGGGYGYYRITYGETGGTCAGCTGGGACGCTCCGGCTGGCGGGGGGTACGGTTATTACCGCATCACTTACGGCGAAACAGGNSPVQEFTVPVSKGTATISGLKAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCPGVDYTITVYAVEFDFPGSGYYCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATTCGACTTCCCGGGCTCCGHRPISINYRTEIDKPSQHHHHHGTTACTACCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCA H (SEQID NO: 211) TCACCACCACCAC (SEQ ID NO: 212) 2382F03MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWDAPAADAYGYYRITYGETGGTCAGCTGGGACGCTCCGGCTGCCGATGCTTACGGTTATTACCGCATCACTTACGGCGAAACAGGNSPVQEFTVPVSKGTATISGLKAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCPGVDYTITVYAVEFNFPGAGYYCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATTCAACTTCCCGGGCGCCGHRPISINYRTEIDKPSQHHHHHGTTACTACCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCA H (SEQID NO: 213) TCACCACCACCAC (SEQ ID NO: 214) 2382F05MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWDAPAEAGKHYGYYRITYGETTCAGCTGGGACGCTCCGGCTGAAGCAGGTAAGCACTACGGTTATTACCGCATCACTTACGGCGAGGNSPVQEFTVPVSKGTATISGAACAGGAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCLKPGVDYTITVYAVEFDFPGAGAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATTCGACTTCCCGGYYHRPISINYRTEIDKPSQHHHGCGCCGGTTACTACCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCA HHH(SEQ ID NO: 215) GCACCATCACCACCACCAC (SEQ ID NO: 216) 2382F08MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWDAPAEAYGYYRITYGETGGNTCAGCTGGGACGCTCCGGCTGAAGCATACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGSPVQEFTVPVSKGTATISGLKPCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCCTTGVDYTITVYAVEFTYPGSGYYHAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATTCACCTACCCCGGCTCCGGTTRPISINYRTEIDKPSQHHHHHHACTACCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCA (SEQ IDNO: 217) CCACCACCAC (SEQ ID NO: 218) 2382F09 MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWDAPAAAYGYYRITYGETGGNTCAGCTGGGACGCTCCGGCTGCAGCCTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGSPVQEFTVPVSKGTATISGLKPCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCCTTGVDYTITVYAVEYDFPGSGYYHAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATACGACTTCCCCGGCTCCGGTTRPISINYRTEIDKPSQHHHHHHACTACCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCA (SEQ IDNO: 219) CCACCACCAC (SEQ ID NO: 220) 2382G04 MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWDAPAGGGYGYYRITYGETGGTCAGCTGGGACGCTCCGGCTGGTGGGGGATACGGTTATTACCGCATCACTTACGGCGAAACAGGNSPVQEFTVPSSKGTATISGLKAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTAGTTCTAAAGGTACAGCTACCATCAGCGGCPGVDYTITVYAVEFDFPGAGYYCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATTCGACTTCCCGGGCGCCGHRPISINYRTEIDKPSQHHHHHGTTACTACCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCA H (SEQID NO: 221) TCACCACCACCAC (SEQ ID NO: 222) 2382H10MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWDAPAGGYGYYRITYGETGGNTCAGCTGGGACGCTCCGGCTGGGGGCTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGSPVQEFTVPVSKGTATISGLKPCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCCTTGVDYTITVYAVEFDFPGSGYYHAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATTCGACTTCCCCGGCTCCGGTTRPISINYRTEIDKPSQHHHHHHACTACCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCA (SEQ IDNO: 223) CCACCACCAC (SEQ ID NO: 224) 2382H11 MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWDAPADGYGYYRITYGETGGNTCAGCTGGGACGCTCCGGCTGATGGTTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGSPVQEFTVPVFKGTATISGLKPCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTTTAAAGGTACAGCTACCATCAGCGGCCTTGVDYTITVYAVEFDYPGSGYYHAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATTCGACTACCCCGGCTCCGGTTRPISINYRTEIDKPSQHHHHHHACTACCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCA (SEQ IDNO: 225) CCACCACCAC (SEQ ID NO: 226) 2382H04 MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWDAPAAGGYGYYRITYGETGGTCAGCTGGGACGCTCCGGCTGCGGGGGGGTACGGTTATTACCGCATCACTTACGGCGAAACAGGNSPVQEFTVPSSKGTATISGLKAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTAGTTCTAAAGGTACAGCTACCATCAGCGGCPGVDYTITVYAVEYDFPGAGYYCTTAAACCTGGCGTTGATTATACCATCACTGTATATGCTGTCGAATACGACTTCCCCGGCGCCGHRPISINYRTEIDKPSQHHHHHGTTACTACCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCA H (SEQID NO: 227) TCACCACCACCAC (SEQ ID NO: 228) 2382H07MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWDAPADAYGYYRITYGETGGNTCAGCTGGGACGCTCCGGCTGATGCTTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGSPVQEFTVPGSKGTATISGLKPCAATAGCCCAGTCCAGGAGTTCACTGTGCCTGGTTCTAAAGGTACAGCTACCATCAGCGGCCTTGVDYTITVYAVEFDFPGSGYYHAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATTCGACTTCCCCGGCTCCGGTTRPISINYRTEIDKPSQHHHHHHACTACCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCA (SEQ IDNO: 229) CCACCACCAC (SEQ ID NO: 230) 2382H09 MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWDAPAAAYGYYRITYGETGGNTCAGCTGGGACGCTCCGGCTGCGGCTTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGSPVQEFTVPSSKGTATISGLKPCAATAGCCCTGTCCAGGAGTTCACTGTGCCTAGTTCTAAAGGTACAGCTACCATCAGCGGCCTTGVDYTITVYAVEFDFPGSGYYHAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATTCGACTTCCCCGGCTCCGGTTRPISINYRTEIDKPSQHHHHHHACTACCATCGCCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCA (SEQ IDNO: 231) CCACCACCAC (SEQ ID NO: 232) 2451A02 MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWDAPAAGYGYYRITYGETGGNTCAGCTGGGACGCTCCGGCTGCGGGTTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGSPVQEFTVPVSKGTATISGLKPCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCCTTGVDYTITVYAVEFPFPGSGYYHAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATTCCCCTTCCCCGGCTCCGGTTRPISINYRTEIDKPSQHHHHHHACTACCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCA (SEQ IDNO: 233) CCACCACCAC (SEQ ID NO: 234) 2451B05 MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWDAPAGGYGYYRITYGETGGNTCAGCTGGGACGCTCCGGCTGGGGGATACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGSPVQEFTVPSSKGTATISGLKPCAATAGCCCTGTCCAGGAGTTCACTGTGCCTAGTTCTAAAGGTACAGCTACCATCAGCGGCCTTGVDYTITVYAVEFDYPGSGYYHAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATTCGACTACCCCGGCTCCGGTTRPISINYRTEIDKPSQHHHHHHACTACCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCA (SEQ IDNO: 235) CCACCACCAC (SEQ ID NO: 236) 2451B06 MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGA(equivalent to SWDAPADGGYGYYRITYGETGGTCAGCTGGGACGCTCCGGCTGATGGTGGTTACGGTTATTACCGCATCACTTACGGCGAAACAGG2382D05) NSPVQEFTVPVSKGTATISGLKAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCPGVDYTITVYAVEFDFPGAGYYCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATTCGACTTCCCGGGCGCCGHRPISINYRTEIDKPSQHHHHHGTTACTACCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCA H (SEQID NO: 205) TCACCACCACCAC (SEQ ID NO: 206) 2451C06MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWDAPAGAASYGYYRITYGETGTCAGCTGGGACGCTCCGGCTGGGGCAGCGTCCTACGGTTATTACCGCATCACTTACGGCGAAACGNSPVQEFTVPVSKGTATISGLAGGAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCKPGVDYTITVYAVEFPFPGAGYGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATTCCCCTTCCCCGGCGYHRPISINYRTEIDKPSQHHHHCCGGTTACTACCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCA HH (SEQID NO: 237) CCATCACCACCACCAC (SEQ ID NO: 238) 2451D05MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWDAPAGAYGYYRITYGETGGNTCAGCTGGGACGCTCCGGCTGGCGCGTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGSPVQEFTVPVSKGTATISGLKPCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCCTTGVDYTITVYAVEFDFPGSGYYHAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATTCGACTTCCCCGGCTCCGGTTRPISINYRTEIDKPSQHHHHHHACTACCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCA (SEQ IDNO: 239) CCACCACCAC (SEQ ID NO: 240) 2451F03 MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWDPPAEGYGYYRITYGETGGNTCAGCTGGGACCCTCCGGCTGAAGGTTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGSPVQEFTVPVSKGTATISGLKPCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCCTTGVDYTITVYAVEFNFPGSGYYHAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATTCAACTTCCCCGGCTCCGGTTRPISINYRTEIDKPSQHHHHHHACTACCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCA (SEQ IDNO: 241) CCACCACCAC (SEQ ID NO: 242) 2451G01 MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWDAPAGGYGYYRITYGETGGNTCAGCTGGGACGCTCCGGCTGGGGGCTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGSPVQEFTVPSSKGTATISGLKPCAATAGCCCTGTCCAGGAGTTCACTGTGCCTAGTTCTAAAGGTACAGCTACCATCAGCGGCCTTGVDYTITVYAVEFDFPGSGYYHAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATTCGACTTCCCGGGCTCCGGTTRPISINYRTEIDKPSQHHHHHHACTACCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCA (SEQ IDNO: 243) CCACCACCAC (SEQ ID NO: 244) 2451H07 MGITDVPRDLEVVAATPTSLLIATGGGTATCACGGATGTGCCGCGAGACTTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWNPPDVNYGYYRITYGETGGNTCAGCTGGAACCCGCCGGATGTGAATTACGGTTATTATCGCATCACTTACGGGGAAACAGGAGGSPLQEFTVPVSKGTATISGLKPCAATAGCCCTTTGCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCCTTGVDYTITVYAVEYPYAHAGYYHAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATATCCGTACGCGCACGCTGGTTRPISINYRTEIDKPSQHHHHHHACTACCATCGTCCGATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCA (SEQ IDNO: 245) CCACCACCAC (SEQ ID NO: 246) 2382E03 MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWDAPAGDGYGYYRITYGETGGTCAGCTGGGACGCTCCGGCTGGGGACGGGTACGGTTATTACCGCATCACTTACGGCGAAACAGGNSPVQEFTVPVSKGTATISGLKAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCPGVDYTITVYAVEFDFPGAGYYCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATTCGACTTCCCCGGCGCCGHRPISINYRTEIDKPSQHHHHHGTTACTACCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCA H (SEQID NO: 247) TCACCACCACCAC (SEQ ID NO: 248) 2382E04MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWDAPAGGGYGYYRITYGETGGTCAGCTGGGACGCTCCGGCTGGTGGTGGATACGGTTATTACCGCATCACTTACGGCGAAACAGGNSPVQEFTVPVSKGTATISGLKAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCPGVDYTITVYAVEFTFPGAGYYCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATTCACCTTCCCGGGCGCCGHRPISINYRTEIDKPSQHHHHHGTTACTACCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCA H (SEQID NO: 249) TCACCACCACCAC (SEQ ID NO: 250) 2382E05MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWDAPAEGGYGYYRITYGETGGTCAGCTGGGACGCTCCGGCTGAGGGCGGCTACGGTTATTACCGCATCACTTACGGCGAAACAGGNSPVQEFTVPVSKGTATISGLKAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCPGVDYTITVYAVEFDFPGAGYYCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATTCGACTTCCCCGGCGCCGHRPISINYRTEIDKPSQHHHHHGTTACTACCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCA H (SEQID NO: 251) TCACCACCACCAC (SEQ ID NO: 252) 2382E09MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWDAPAEAYGYYRITYGETGGNTCAGCTGGGACGCTCCGGCTGAGGCTTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGSPVQEFTVPVSKGTATISGLKPCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCCTTGVDYTITVYAVEYDFPGSGYYHAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATACGACTTCCCCGGCTCCGGTTRPISINYRTEIDKPSQHHHHHHACTACCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCA (SEQ IDNO: 253) CCACCACCAC (SEQ ID NO: 254) 2381A04 MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWEPFSRLPGGGEYYRITYGETTCAGCTGGGAGCCGTTCAGCCGGTTGCCCGGGGGCGGCGAGTATTACCGGATCACTTACGGCGAGGNSPLQQFTVPGSKGTATISGAACAGGAGGCAATAGCCCTCTGCAGCAGTTCACTGTGCCTGGTTCTAAAGGTACAGCTACCATCLKPGVDYTITVYAVEYPYPFSGAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATACCCGTACCCGTYYHRPISINYRTEIDKPSQHHHTTTCTGGTTACTACCATCGTCCCATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCA HHH(SEQ ID NO: 255) GCACCATCACCACCACCAC (SEQ ID NO: 256) 2381A08MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWDAPADGGYGYYRITYGETGGTCAGCTGGGACGCTCCGGCTGACGGCGGGTACGGTTATTACCGCATCACTTACGGCGAAACAGGNSPVQEFTVPGSKGTATISGLKAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGGTTCTAAAGGTACAGCTACCATCAGCGGCPGVDYTITVYAVEYDFPGAGYYCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATACGACTTCCCGGGCGCCGHRPISINYRTEIDKPSQHHHHHGTTACTACCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCA H (SEQID NO: 257) TCACCACCACCAC (SEQ ID NO: 258) 2381B10MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWDAPAGGGYGYYRITYGETGGTCAGCTGGGACGCTCCGGCTGGGGGTGGATACGGTTATTACCGSATCACTTACGGCGAAACAGGNSPVQEFTVPVSKGTATISGLKAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCPGVDYTITVYAVEYNFIGAGYYCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATACAACTTCATCGGCGCCGHRPISINYRTEIDKPSQHHHHHGTTACTACCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCA H (SEQID NO: 259) TCACCACCACCAC (SEQ ID NO: 260) 2381C08MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWDAPADGAYGYYRITYGETGGTCAGCTGGGACGCTCCGGCTGACGGTGCCTACGGTTATTACCGCATCACTTACGGCGAAACAGGNSPVQEFTVPVSKGTATISGLKAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCPGVDYTITVYAVEFPYPFAGYYCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATTCCCCTACCCCTTCGCCGHRPISINYRTEIDKPSQHHHHHGTTACTACCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCA H (SEQID NO: 261) TCACCACCACCAC (SEQ ID NO: 262) 2381G06MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWSEKLDGKARRGYYRITYGETTCAGCTGGTCGGAGAAGTTGGACGGGAAGGCGCGCCGCGGGTATTACCGCATCACATACGGCGAGGNSPVQQFTVPGSKGTATISGAACAGGAGGCAATAGCCCTGTCCAGCAGTTCACTGTGCCTGGTTCTAAAGGTACAGCTACCATCLKPGVDYTITVYAVEFPYDHSGAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATTCCCGTACGACCYYHRPISINYRTEIDKPSQHHHATTCTGGTTACTACCATCGTCCCATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCA HHH(SEQ ID NO: 263) GCACCATCACCACCACCAC (SEQ ID NO: 264) 2381H01MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWSPRDSTGLVRRGYYRITYGETCAGCTGGAGCCCGCGGGACTCCACCGGCTTGGTGAGGCGGGGGTATTACCGCATCACTTACGGTGGNSPVQQFTVPGSKGTATISCGAAACAGGAGGCAATAGCCCTGTTCAGCAGTTCACTGTGCCTGGTTCTAAAGGTACAGCTACCGLKPGVDYTITVYAVEYPYDHSATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATACCCGTACGGYYHRPISINYRTEIDKPSQHHACCATTCTGGTTACTACCATCGGCCCATTTCCATTAATTACCGCACAGAAATTGACAAACCATC HHHH(SEQ ID NO: 265) CCAGCACCATCACCACCACCAC (SEQ ID NO: 266) 2381H06MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWGDVRTNEARQGYYRITYGETTCAGCTGGGGCGACGTCCGGACGAACGAGGCGCGGCAGGGCTATTACCGGATCACTTACGGCGAGGNSPLQGFTVPGSKGTATISGAACAGGAGGCAATAGCCCTCTCCAGGGGTTCACTGTGCCTGGTTCTAAAGGTACAGCTACCATCLKPGVDYTITVYAVEYTYEHSGAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAGTATACGTACGAGCYYHRPISINYRTEIDKPSQHHHATTCTGGTTACTACCATCGTCCGATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCA HHH(SEQ ID NO: 267) GCACCATCACCACCACCAC (SEQ ID NO: 268) 2381H09MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWDAPAGGGYGYYRITYGETGGTCAGCTGGGACGCTCCGGCTGGGGGGGGCTACGGTTATTACCGCATCACTTACGGCGAAACAGGNSPVQEFTVPVSKGTATISGLKAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCPGVDYTITVYAVEFDFVGAGYYCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATTCGACTTCGTCGGCGCCGHRPISINYRTEIDKPSQHHHHHGTTACTACCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCA H (SEQID NO: 269) TCACCACCACCAC (SEQ ID NO: 270) 2382B11MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWDAPAAAYGYYRITYGETGGNTCAGCTGGGACGCTCCGGCTGCGGCCTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGSPVQEFTVPVSKGTATISGLKPCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCCTTGVDYTITVYAVEYDFAGSGYYHAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATACGACTTCGCGGGCTCCGGTTRPISINYRTEIDKPSQHHHHHHACTACCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCA (SEQ IDNO: 271) CCACCACCAC (SEQ ID NO: 272) 2382B08 MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWDAPADAYGYYRITYGETGGNTCAGCTGGGACGCTCCGGCTGACGCGTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGSPVQEFTVPSSKGTATISGLKPCAATAGCCCTGTCCAGGAGTTCACTGTGCCTAGTTCTAAAGGTACAGCTACCATCAGCGGCCTTGVDYTITVYAVEFAFPGAGYYHAAACCTGGCGTTGATTATACCATCACTGTATATGCTGTCGAATTCGCCTTCCCCGGCGCCGGTTRPISINYRTEIDKPSQHHHHHHACTACCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCA (SEQ IDNO: 273) CCACCACCAC (SEQ ID NO: 274) 2382C11 MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWDAPAGGYGYYRITYGETGGNTCAGCTGGGACGCTCCGGCTGGAGGTTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGSPVQEFTVPVSKGTATISGLKPCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCCTTGVDYTITVYAVEYDFAGSGYYHAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATACGACTTCGCGGGCTCCGGTTRPISINYRTEIDKPSQHHHHHHACTACCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCA (SEQ IDNO: 275) CCACCACCAC (SEQ ID NO: 276) 2382G03 MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWDAPAEAEAYGYYRITYGETGTCAGCTGGGACGCTCCGGCTGAAGCAGAAGCGTACGGTTATTACCGCATCACTTACGGCGAAACGNSPVQEFTVPVSKGTATISGLAGGAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCKPGVDYTITVYAVEYVFPGAGYGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATACGTCTTCCCCGGCGYHRPISINYRTEIDKPSQHHHHCCGGTTACTACCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCA HH (SEQID NO: 277) CCATCACCACCACCAC (SEQ ID NO: 278) 2382H03MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWDAPAEGAYGYYRITYGETGGTCAGCTGGGACGCTCCGGCTGAGGGCGCTTACGGTTATTACCGCATCACTTACGGCGAAACAGGNSPVQEFTVPVSKGTATISGLKAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCPGVDYTITVYAVEYPYPFAGYYCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATACCCCTACCCCTTCGCCGHRPISINYRTEIDKPSQHHHHHGTTACTACCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCA H (SEQID NO: 279) TCACCACCACCAC (SEQ ID NO: 280) 2451A10MGVTDVPRDMEVVAATPTSLLIATGGGTGTCACCGATGTGCCGCGCGACATGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWQPPAVTYGYYRITYGETGGNTCAGCTGGCAGCCGCCGGCTGTTACTTACGGTTATTATCGCATCACTTACGGCGAAACAGGAGGSTLQQFTVPVYKGTATISGLKPCAATAGCACTCTCCAGCAGTTCACTGTGCCTGTTTATAAAGGTACAGCTACCATCAGCGGCCTTGVDYTITVYAVEYPYDHSGYYHAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATACCCGTACGACCATTCTGGGTRPISINYRTEIDKPSQHHHHHHACTACCATCGGCCGATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCA (SEQ IDNO: 281) CCACCACCAC (SEQ ID NO: 282) 2451B02 MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWDAPAAAYGYYRITYGETGGNTCAGCTGGGACGCTCCGGCTGCTGCTTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGSPVQEFTVPVSKGTATISGLKPCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCCTTGVDYTITVYAVEFDYPGSGYYHAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATTCGACTACCCCGGCTCCGGTTRPISINYRTEIDKPSQHHHHHHACTACCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCA (SEQ IDNO: 283) CCACCACCAC (SEQ ID NO: 284) 2451C11 MGIVDVPRDLEVVAATPTSLLIATGGGTATCGTGGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWDPPAGAYGYYRITYGETGGNTCAGCTGGGACCCGCCGGCTGGTGCTTACGGTTATTATCGCATCACTTACGGCGAAACAGGAGGSPKQQFTVPGYKGTATISGLKPCAATAGCCCAAAGCAGCAGTTCACTGTGCCTGGTTATAAAGGTACAGCTACCATCAGCGGCCTTGVDYTITVYAVEYPYDHSGYYHAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATACCCGTACGACCATTCTGGTTRPISINYRTEIDKPSQHHHHHHACTACCATCGGCCGATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCA (SEQ IDNO: 285) CCACCACCAC (SEQ ID NO: 286) 2451H01 MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWDAPAAGYGYYRITYGETGGNTCAGCTGGGACGCTCCGGCTGCGGGGTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGSPVQEFTVPVSKGTATISGLKPCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCCTTGVDYTITVYAVEYDFPGSGYYHAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATACGACTTCCCCGGCTCCGGTTRPISINYRTEIDKPSQHHHHHHACTACCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCA (SEQ IDNO: 287) CCACCACCAC (SEQ ID NO: 288) 2011B11 MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWAPPSDAYGYYRITYGETGGNTCAGCTGGGCGCCGCCTTCTGATGCGTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGSPVQEFTVPIGKGTATISGLKPCAATAGCCCTGTCCAGGAGTTCACTGTGCCTATTGGTAAAGGTACAGCTACCATCAGCGGCCTTGVDYTITVYAVEYPYSHAGYYHAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATACCCGTATTCACATGCTGGTTRPISINYRTEIDKPSQHHHHHHACTACCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCA (SEQ IDNO: 289) CCACCACCAC (SEQ ID NO: 290) 2971A03 MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWDPPSDDYGYYRITYGETGGNTCAGCTGGGACCCGCCTTCGGATGATTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGSPVQEFTVPIGKGTATISGLKPCAATAGCCCTGTCCAGGAGTTCACTGTGCCTATTGGTAAAGGAACAGCTACCATCAGCGGCCTTGVDYTITVYAVEFPWPHAGYYHAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAGTTTCCGTGGCCACATGCTGGTTRPISINYRTEIDKPSQHHHHHHACTATCATCGGCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCA (SEQ IDNO: 304) CCACCACCAC (SEQ ID NO: 305) 2971A09 MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWDAPADDYGYYRITYGETGGNTCAGCTGGGACGCGCCTGCGGATGATTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGSPVQEFTVPIGKGTATISGLKPCAATAGCCCTGTCCAGGAGTTCACTGTGCCTATTGGTAAAGGAACAGCTACCATCAGCGGCCTTGVDYTITVYAVEFPWPHAGYYHAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAGTTTCCGTGGCCACATGCTGGTTRPISINYRTEIDKPSQHHHHHHACTATCATCGGCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCA (SEQ IDNO: 306) CCACCACCAC (SEQ ID NO: 307) 2971E02 MGVSDVPRDLEVVAATPTSLLIATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGASWDAPSDDYGYYRITYGETGGNTCAGCTGGGACGCGCCTTCGGATGATTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGSPVQEFTVPIGKGTATISGLKPCAATAGCCCTGTCCAGGAGTTCACTGTGCCTATTGGTAAAGGAACAGCTACCATCAGCGGCCTTGVDYTITVYAVEFPWPHAGYYHAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAGTTTCCGTGGCCACATGCTGGTTRPISINYRTEIDKPSQHHHHHHACTATCATCGGCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCA (SEQ IDNO: 308) CCACCACCAC (SEQ ID NO: 309)

The SEQ ID NOS of the family of the pegylated anti-PCSK9 Adnectins ofthe invention are presented in Table 5.

TABLE 5 Anti-PCSK9 Adnectin Family Cysteine Mutants to Enable PegylationATI#/Clone# Sequence [Description] AA NT ATI001170 MGVSDVPRDLEVVAATPTSLATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGAT[2013E01-non His LISWVPPSDDYGYYRITYGECAGCTGGGTCCCGCCTTCAGATGATTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCA taggedCys mut] TGGNSPVQEFTVPIGKGTATATAGCCCTGTCCAGGAGTTCACTGTGCCTATTGGTAAAGGAACAGCTACCATCAGCGGCCTTAAAISGLKPGVDYTITVYAVEFPCCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAGTTTCCGTGGCCACATGCTGGTTACTAWPHAGYYHRPISINYRTEIDTCATCGGCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATGCCAGTG (SEQ ID KPCQ (SEQID NO: 78) NO: 79) ATI001172 MGVSDVPRDLEVVAATPTSLATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGAT[2013E01-non His LISWVPPSDDYGYYRITYGECAGCTGGGTCCCGCCTTCAGATGATTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCA taggedCys mut] TGGNSPVQEFTVPIGKGTATATAGCCCTGTCCAGGAGTTCACTGTGCCTATTGGTAAAGGAACAGCTACCATCAGCGGCCTTAAAISGLKPGVDYTITVYAVEFPCCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAGTTTCCGTGGCCACATGCTGGTTACTAWPHAGYYHRPISINYRTEGS TCATCGGCCAATTTCCATTAATTACCGAACAGAAGGTAGCGGTTGCTG(SEQ ID NO: 81) GC (SEQ ID NO: 80) ATI001174* MGVSDVPRDLEVVAATPTSLATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGAT[2013E01-non His LISWVPPSDDYGYYRITYGECAGCTGGGTCCCGCCTTCAGATGATTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCA taggedCys mut] TGGNSPVQEFTVPIGKGTATATAGCCCTGTCCAGGAGTTCACTGTGCCTATTGGTAAAGGAACAGCTACCATCAGCGGCCTTAAA alsoreferred ISGLKPGVDYTITVYAVEFPCCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAGTTTCCGTGGCCACATGCTGGTTACTA to asATI-1174 WPHAGYYHRPISINYRTEIETCATCGGCCAATTTCCATTAATTACCGCACAGAAATTGAGAAACCATGCCAGTG (SEQ ID KPCQ (SEQID NO: 82) NO: 83) ATI001114* MGVSDVPRDLEVVAATPTSLATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGAT[2013E01cys mut] LISWVPPSDDYGYYRITYGECAGCTGGGTCCCGCCTTCAGATGATTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCA alsoreferred TGGNSPVQEFTVPIGKGTATATAGCCCTGTCCAGGAGTTCACTGTGCCTATTGGTAAAGGAACAGCTACCATCAGCGGCCTTAAA to asATI-1114 ISGLKPGVDYTITVYAVEFPCCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAGTTTCCGTGGCCACATGCTGGTTACTAWPHAGYYHRPISINYRTGSGTCATCGGCCAATTTCCATTAATTACCGCACAGGTAGCGGTTGCCACCATCACCACCATCAC CHHHHHH(SEQ ID (SEQ ID NO: 85) NO: 84) ATI000959* MGVSDVPRDLEVVAATPTSLATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGAT[1459D05 cys LISWPPPSHGYGYYRITYGECAGCTGGCCGCCGCCGTCTCATGGTTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCA mut]TGGNSPVQEFTVPPGKGTATATAGCCCTGTCCAGGAGTTCACTGTGCCGCCTGGTAAAGGTACAGCTACCATCAGCGGCCTTAAAISGLKPGVDYTITVYAVEYPCCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATACCCGTACAAACATTCTGGTTACTAYKHSGYYHRPISINYRTEIDCCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATGCCAGCACCATCACCACCKPCQHHHHHH (SEQ ID ACCAC (SEQ ID NO: 87) NO: 86) ATI001063*MGVSDVPRDLEVVAATPTSLATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGAT[1784F03 Cys LISWRPPIHAYGYYRITYGECAGCTGGAGGCCGCCGATTCATGCTTACGGGTATTACCGCATCACTTACGGCGAAACAGGAGGCA mut]TGGNSPVQEFTVPIVEGTATATAGCCCTGTCCAGGAGTTCACTGTGCCTATTGTTGAAGGTACAGCTACCATCAGCGGCCTTAAAISGLKPGVDYTITVYAVEYTCCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATATACATTTAAACATTCCGGTTACTAFKHSGYYHRPISINYRTEIDCCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATGCCAGCACCATCACCACCKPCQHHHHHH (SEQ ID ACCAC (SEQ ID NO: 89) NO: 88) ATI001119*MGVSDVPRDLEVVAATPTSLATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGAT[2012A04 Cys LISWRPPSNGHGYYRITYGECAGCTGGCGGCCCCCCTCTAATGGTCACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCA mut]TGGNSPVQEFTVPVNEGTATATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTAATGAAGGTACAGCTACCATCAGCGGCCTTAAAISGLKPGVDYTITVYAVEFPCCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATTCCCCTTCAAGTGGTCGGGCTACTAFKWSGYYHRPISINYRTGSGCCATCGACCAATTTCCATTAATTACCGCACAGGTAGCGGTTGCCACCATCACCACCATCAC CHHHHHH(SEQ ID (SEQ ID NO: 91) NO: 90) ATI001117* MGVSDVPRDLEVVAATPTSLATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGAT[2011H05 Cys LISWVPSSHAYGYYRITYGECAGCTGGGTTCCGTCGTCTCATGCCTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCA mut]TGGNSPVQEFTVPVGVGTATATAGCCCTGTCCAGGAGTTCACTGTGCCTGTGGGGGTAGGTACAGCTACCATCAGCGGCCTTAAAISGLKPGVDYTITVYAVEYACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATACGCTTTCGAAGGGGCTGGTTACTAFEGAGYYHRPISINYRTGSGCCATCGTCCAATTTCCATTAATTACCGCACAGGTAGCGGTTGCCACCATCACCACCATCAC CHHHHHH(SEQ ID (SEQ ID NO: 93) NO: 92) ATI001194* MGVSDVPRDLEVVAATPTSLATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGAT[2011H05(V23D) LISWDPSSHAYGYYRITYGECAGCTGGGACCCGTCGTCTCATGCCTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCA Cysmut] TGGNSPVQEFTVPVGVGTATATAGCCCTGTCCAGGAGTTCACTGTGCCTGTGGGGGTAGGTACAGCTACCATCAGCGGCCTTAAAISGLKPGVDYTITVYAVEYACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATACGCTTTCGAAGGGGCTGGTTACTAFEGAGYYHRPISINYRTEGSCCATCGTCCAATTTCCATTAATTACCGCACAGAAGGTAGCGGTTGCCACCATCACCACCATCACGCHHHHHH (SEQ ID (SEQ ID NO: 95) NO: 94) 2011H05 (V23E)-MGVSDVPRDLEVVAATPTSLATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGAT Cysmut LISWEPSSHAYGYYRITYGECAGCTGGGAACCGTCGTCTCATGCCTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCATGGNSPVQEFTVPVGVGTATATAGCCCTGTCCAGGAGTTCACTGTGCCTGTGGGGGTAGGTACAGCTACCATCAGCGGCCTTAAAISGLKPGVDYTITVYAVEYACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATACGCTTTCGAAGGGGCTGGTTACTAFEGAGYYHRPISINYRTEGSCCATCGTCCAATTTCCATTAATTACCGCACAGAAGGTAGCGGTTGCCACCATCACCACCATCACGCHHHHHH (SEQ ID (SEQ ID NO: 97) NO: 96) ATI001112 MGVSDVPRDLEVVAATPTSLATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGAT[1923B02 Cys LISWTPPPKGYGYYRITYGECAGCTGGACGCCTCCCCCTAAAGGGTATGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCA mut]TGGNSPVQEFTVPVGEGTATATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTGGTGAAGGTACAGCTACCATCAGCGGCCTTAAAISGLKPGVDYTITVYAVEYTCCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATACACGTACAACGGTGCCGGTTACTAYNGAGYYHRPISINYRTGSGCCACCGGCCAATTTCCATTAATTACCGCACAGGTAGCGGTTGCCACCATCACCACCATCAC CHHHHHH(SEQ ID (SEQ ID NO: 99) NO: 98) ATI001110 MGVSDVPRDLEVVAATPTSLATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGAT[1922G04 Cys LISWRPPSHAYGYYRITYGECAGCTGGCGGCCGCCATCTCATGCTTATGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCA mut]TGGNSPVQEFTVPIGKGTATATAGCCCTGTCCAGGAGTTCACTGTGCCTATTGGGAAAGGTACAGCTACCATCAGCGGCCTTAAAISGLKPGVDYTITVYAVEYPCCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATACCCGTGGAAAGGTTCTGGTTACTAWKGSGYYHRPISINYRTGSGCCATCGGCCAATTTCCATTAATTACCGCACAGGTAGCGGTTGCCACCATCACCACCATCAC CHHHHHH(SEQ ID (SEQ ID NO: 101) NO: 100) ATI001128 MGVSDVPRDLEVVAATPTSLATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGAT[1922G04 Cys LISWRPPSHAYGYYRITYGECAGCTGGCGGCCGCCATCTCATGCTTATGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCA mut]TGGNSPVQEFTVPIGKGTATATAGCCCTGTCCAGGAGTTCACTGTGCCTATTGGGAAAGGTACAGCTACCATCAGCGGCCTTAAAISGLKPGVDYTITVYAVEYPCCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATACCCGTGGAAAGGTTCTGGTTACTAWKGSGYYHRPISINYRTEIDCCATCGGCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATGCCAGCACCACCACCACCKPCQHHHHHH (SEQ ID ACCAC (SEQ ID NO: 103) NO: 102) ATI001184 *MGVSDVPRDLEVVAATPTSLATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGAT[1922G04(R23E) LISWEPPSHAYGYYRITYGECAGCTGGGAACCGCCATCTCATGCTTATGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCA Cysmut] TGGNSPVQEFTVPIGKGTATATAGCCCTGTCCAGGAGTTCACTGTGCCTATTGGGAAAGGTACAGCTACCATCAGCGGCCTTAAAISGLKPGVDYTITVYAVEYPCCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATACCCGTGGAAAGGTTCTGGTTACTAWKGSGYYHRPISINYRTEGSCCATCGGCCAATTTCCATTAATTACCGCACAGAAGGTAGCGGTTGCCACCATCACCACCATCACGCHHHHHH (SEQ ID (SEQ ID NO: 105) NO: 104) 2381D04-CysMGVSDVPRDLEVVAATPTSLATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGATLISWEPFSRLPGGGEYYRITCAGCTGGGAGCCGTTCAGCCGGTTGCCCGGGGGCGGCGAGTATTACCGGATCACTTACGGCGAAAYGETGGNSPLQQFTVPGSKGCAGGAGGCAATAGCCCTCTGCAGCAGTTCACTGTGCCTGGTTCTAAAGGTACAGCTACCATCAGCTATISGLKPGVDYTITVYAVGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATTCCCGTACGACCATTCEFPYDHSGYYHRPISINYRTTGGTTACTACCATCGGCCCATTTCCATTAATTACCGCACAGGTAGCGGTTGCCACCATCACCACCGSGCHHHHHH (SEQ ID ATCAC (SEQ ID NO: 292) NO: 291) 2382D09-CysMGVSDVPRDLEVVAATPTSLATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGATLISWDAPAEGYGYYRITYGECAGCTGGGACGCTCCGGCTGAAGGGTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCATGGNSPVQEFTVPVSKGTATATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCCTTAAAISGLKPGVDYTITVYAVEFDCCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATTCGACTTCCCCGGCGCCGGTTACTAFPGAGYYHRPISINYRTGSGCCATCGTCCAATTTCCATTAATTACCGCACAGGTAGCGGTTGCCACCATCACCACCATCAC CHHHHHH(SEQ ID (SEQ ID NO: 294) NO: 293) 2451B06-Cys MGVSDVPRDLEVVAATPTSLATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGATLISWDAPADGGYGYYRITYGCAGCTGGGACGCTCCGGCTGATGGTGGTTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGETGGNSPVQEFTVPVSKGTAGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCCTTTISGLKPGVDYTITVYAVEFAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATTCGACTTCCCGGGCGCCGGTTADFPGAGYYHRPISINYRTGSCTACCATCGTCCAATTTCCATTAATTACCGCACAGGTAGCGGTTGCCACCATCACCACCATCACGCHHHHHH (SEQ ID (SEQ ID NO: 296) NO: 295) 2382E05-CysMGVSDVPRDLEVVAATPTSLATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGATLISWDAPAEGGYGYYRITYGCAGCTGGGACGCTCCGGCTGAGGGCGGCTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGETGGNSPVQEFTVPVSKGTAGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCCTTTISGLKPGVDYTITVYAVEFAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATTCGACTTCCCCGGCGCCGGTTADFPGAGYYHRPISINYRTGSCTACCATCGTCCAATTTCCATTAATTACCGCACAGGTAGCGGTTGCCACCATCACCACCATCACGCHHHHHH (SEQ ID (SEQ ID NO: 298) NO: 297) 2382B09-CysMGVSDVPRDLEVVAATPTSLATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGATLISWDAPADAYGYYRITYGECAGCTGGGACGCTCCGGCTGACGCGTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCATGGNSPVQEFTVPVSKGTATATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCCTTAAAISGLKPGVDYTITVYAVEFDCCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATTCGACTACCCCGGCTCCGGTTACTAYPGSGYYHRPISINYRTGSGCCATCGTCCAATTTCCATTAATTACCGCACAGGTAGCGGTTGCCACCATCACCACCATCAC CHHHHHH(SEQ ID (SEQ ID NO: 300) NO: 299) *Note: Some proteins listed have notyet been pegylated but are enabled to be pegylated via the cysteinemutation. Proteins that have been pegylated are indicated by asterisk.Nucleic Acid-Protein Fusion Technology

In one aspect, the application provides an Adnectin comprisingfibronectin type III domains that binds PCSK9. One way to rapidly makeand test Fn3 domains with specific binding properties is the nucleicacid-protein fusion technology of Adnexus, a Bristol-Myers Squibb R&DCompany. This disclosure utilizes the in vitro expression and taggingtechnology, termed PROfusion which exploits nucleic acid-protein fusions(RNA- and DNA-protein fusions) to identify novel polypeptides and aminoacid motifs that are important for binding to proteins. Nucleicacid-protein fusion technology is a technology that covalently couples aprotein to its encoding genetic information. For a detailed descriptionof the RNA-protein fusion technology and fibronectin-based scaffoldprotein library screening methods see Szostak et al., U.S. Pat. Nos.6,258,558, 6,261,804, 6,214,553, 6,281,344, 6,207,446, 6,518,018 and6,818,418; and Roberts et al., Proc. Natl. Acad. Sci., 94:12297-12302(1997).

Vectors and Polynucleotide Embodiments

Nucleic acids encoding any of the various proteins or polypeptidesdisclosed herein may be synthesized chemically. Codon usage may beselected so as to improve expression in a cell. Such codon usage willdepend on the cell type selected. Specialized codon usage patterns havebeen developed for E. coli and other bacteria, as well as mammaliancells, plant cells, yeast cells and insect cells. See for example:Mayfield et al., Proc. Natl. Acad. Sci. USA, 100(2):438-442 (Jan. 21,2003); Sinclair et al., Protein Expr. Purif, 26(I):96-105 (October2002); Connell, N. D., Curr. Opin. Biotechnol., 12(5):446-449 (October2001); Makrides et al., Microbiol. Rev., 60(3):512-538 (September 1996);and Sharp et al., Yeast, 7(7):657-678 (October 1991).

General techniques for nucleic acid manipulation are described forexample in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ndEdition, Vols. 1-3, Cold Spring Harbor Laboratory Press (1989), orAusubel, F. et al., Current Protocols in Molecular Biology, GreenPublishing and Wiley-Interscience, New York (1987) and periodic updates.Generally, the DNA encoding the polypeptide is operably linked tosuitable transcriptional or translational regulatory elements derivedfrom mammalian, viral, or insect genes. Such regulatory elements includea transcriptional promoter, an optional operator sequence to controltranscription, a sequence encoding suitable mRNA ribosomal bindingsites, and sequences that control the termination of transcription andtranslation. The ability to replicate in a host, usually conferred by anorigin of replication, and a selection gene to facilitate recognition oftransformants is additionally incorporated.

The proteins described herein may be produced recombinantly not onlydirectly, but also as a fusion polypeptide with a heterologouspolypeptide, which is preferably a signal sequence or other polypeptidehaving a specific cleavage site at the N-terminus of the mature proteinor polypeptide. The heterologous signal sequence selected preferably isone that is recognized and processed (i.e., cleaved by a signalpeptidase) by the host cell. An exemplary N-terminal leader sequence forproduction of polypeptides in a mammalian system is METDTLLLWVLLLWVPGSTG(SEQ ID NO: 326), which is removed by the host cell followingexpression.

For prokaryotic host cells that do not recognize and process a nativesignal sequence, the signal sequence is substituted by a prokaryoticsignal sequence selected, for example, from the group of the alkalinephosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders.

For yeast secretion the native signal sequence may be substituted by,e.g., the yeast invertase leader, a factor leader (includingSaccharomyces and Kluyveromyces alpha-factor leaders), or acidphosphatase leader, the C. albicans glucoamylase leader, or the signaldescribed in U.S. Pat. No. 5,631,144. In mammalian cell expression,mammalian signal sequences as well as viral secretory leaders, forexample, the herpes simplex gD signal, are available. The DNA for suchprecursor regions may be ligated in reading frame to DNA encoding theprotein.

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2 micron plasmid origin is suitablefor yeast, and various viral origins (SV40, polyoma, adenovirus, VSV orBPV) are useful for cloning vectors in mammalian cells. Generally, theorigin of replication component is not needed for mammalian expressionvectors (the SV40 origin may typically be used only because it containsthe early promoter).

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tracycline, (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media, e.g., the gene encoding D-alanine racemase for Bacilli.

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the nucleicacid encoding the protein of the invention, e.g., a fibronectin-basedscaffold protein. Promoters suitable for use with prokaryotic hostsinclude the phoA promoter, beta-lactamase and lactose promoter systems,alkaline phosphatase, a tryptophan (trp) promoter system, and hybridpromoters such as the tan promoter. However, other known bacterialpromoters are suitable. Promoters for use in bacterial systems also willcontain a Shine-Dalgarno (S.D.) sequence operably linked to the DNAencoding the protein of the invention. Promoter sequences are known foreukaryotes. Virtually all eukaryotic genes have an AT-rich regionlocated approximately 25 to 30 bases upstream from the site wheretranscription is initiated. Another sequence found 70 to 80 basesupstream from the start of transcription of many genes is a CNCAATregion where N may be any nucleotide. At the 3′ end of most eukaryoticgenes is an AATAAA sequence that may be the signal for addition of thepoly A tail to the 3′ end of the coding sequence. All of these sequencesare suitably inserted into eukaryotic expression vectors.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase or other glycolyticenzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Transcription from vectors in mammalian host cells can be controlled,for example, by promoters obtained from the genomes of viruses such aspolyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovinepapilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus,hepatitis-B virus and most preferably Simian Virus 40 (SV40), fromheterologous mammalian promoters, e.g., the actin promoter or animmunoglobulin promoter, from heat-shock promoters, provided suchpromoters are compatible with the host cell systems.

Transcription of a DNA encoding proteins of the invention by highereukaryotes is often increased by inserting an enhancer sequence into thevector. Many enhancer sequences are now known from mammalian genes(globin, elastase, albumin, α-fetoprotein, and insulin). Typically,however, one will use an enhancer from a eukaryotic cell virus. Examplesinclude the SV40 enhancer on the late side of the replication origin (bp100-270), the cytomegalovirus early promoter enhancer, the polyomaenhancer on the late side of the replication origin, and adenovirusenhancers. See also Yaniv, Nature, 297:17-18 (1982) on enhancingelements for activation of eukaryotic promoters. The enhancer may bespliced into the vector at a position 5′ or 3′ to the peptide-encodingsequence, but is preferably located at a site 5′ from the promoter.

Expression vectors used in eukaryotic host cells (e.g., yeast, fungi,insect, plant, animal, human, or nucleated cells from othermulticellular organisms) will also contain sequences necessary for thetermination of transcription and for stabilizing the mRNA. Suchsequences are commonly available from the 5′ and, occasionally 3′,untranslated regions of eukaryotic or viral DNAs or cDNAs. These regionscontain nucleotide segments transcribed as polyadenylated fragments inthe untranslated portion of mRNA encoding the protein of the invention.One useful transcription termination component is the bovine growthhormone polyadenylation region. See WO 94/11026 and the expressionvector disclosed therein.

The recombinant DNA can also include any type of protein tag sequencethat may be useful for purifying the protein. Examples of protein tagsinclude but are not limited to a histidine tag, a FLAG tag, a myc tag,an HA tag, or a GST tag. Appropriate cloning and expression vectors foruse with bacterial, fungal, yeast, and mammalian cellular hosts can befound in Cloning Vectors: A Laboratory Manual, (Elsevier, New York(1985)).

The expression construct is introduced into the host cell using a methodappropriate to the host cell, as will be apparent to one of skill in theart. A variety of methods for introducing nucleic acids into host cellsare known in the art, including, but not limited to, electroporation;transfection employing calcium chloride, rubidium chloride, calciumphosphate, DEAE-dextran, or other substances; microprojectilebombardment; lipofection; and infection (where the vector is aninfectious agent).

Suitable host cells include prokaryotes, yeast, mammalian cells, orbacterial cells. Suitable bacteria include gram negative or grampositive organisms, for example, E. coli or Bacillus spp. Yeast,preferably from the Saccharomyces species, such as S. cerevisiae, mayalso be used for production of polypeptides. Various mammalian or insectcell culture systems can also be employed to express recombinantproteins. Baculovirus systems for production of heterologous proteins ininsect cells are reviewed by Luckow et al. (Bio/Technology, 6:47(1988)). Examples of suitable mammalian host cell lines includeendothelial cells, COS-7 monkey kidney cells, CV-1, L cells, C127, 3T3,Chinese hamster ovary (CHO), human embryonic kidney cells, HeLa, 293,293T, and BHK cell lines. Purified polypeptides are prepared byculturing suitable host/vector systems to express the recombinantproteins. For many applications, the small size of many of thepolypeptides disclosed herein would make expression in E. coli as thepreferred method for expression. The protein is then purified fromculture media or cell extracts.

Protein Production

Host cells are transformed with the herein-described expression orcloning vectors for protein production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.In the examples shown here, the host cells used for high-throughputprotein production (HTPP) and mid-scale production was theHMS174-bacterial strain. The host cells used to produce the proteins ofthis invention may be cultured in a variety of media. Commerciallyavailable media such as Ham's F10 (Sigma), Minimal Essential Medium((MEM), (Sigma)), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle'sMedium ((DMEM), Sigma)) are suitable for culturing the host cells. Inaddition, many of the media described in Ham et al., Meth. Enzymol.,58:44 (1979), Barites et al., Anal. Biochem., 102:255 (1980), U.S. Pat.Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655, 5,122,469, 6,048,728,5,672,502, or U.S. Pat. No. RE 30,985 may be used as culture media forthe host cells. Any of these media may be supplemented as necessary withhormones and/or other growth factors (such as insulin, transferrin, orepidermal growth factor), salts (such as sodium chloride, calcium,magnesium, and phosphate), buffers (such as HEPES), nucleotides (such asadenosine and thymidine), antibiotics (such as Gentamycin drug), traceelements (defined as inorganic compounds usually present at finalconcentrations in the micromolar range), and glucose or an equivalentenergy source. Any other necessary supplements may also be included atappropriate concentrations that would be known to those skilled in theart. The culture conditions, such as temperature, pH, and the like, arethose previously used with the host cell selected for expression, andwill be apparent to the ordinarily skilled artisan.

Proteins disclosed herein can also be produced using cell-translationsystems. For such purposes the nucleic acids encoding the polypeptidemust be modified to allow in vitro transcription to produce mRNA and toallow cell-free translation of the mRNA in the particular cell-freesystem being utilized (eukaryotic such as a mammalian or yeast cell-freetranslation system or prokaryotic such as a bacterial cell-freetranslation system.

Proteins of the invention can also be produced by chemical synthesis(e.g., by the methods described in Solid Phase Peptide Synthesis, 2ndEdition, The Pierce Chemical Co., Rockford, Ill. (1984)). Modificationsto the protein can also be produced by chemical synthesis.

The proteins of the present invention can be purified byisolation/purification methods for proteins generally known in the fieldof protein chemistry. Non-limiting examples include extraction,recrystallization, salting out (e.g., with ammonium sulfate or sodiumsulfate), centrifugation, dialysis, ultrafiltration, adsorptionchromatography, ion exchange chromatography, hydrophobic chromatography,normal phase chromatography, reversed-phase chromatography, getfiltration, gel permeation chromatography, affinity chromatography,electrophoresis, countercurrant distribution or any combinations ofthese. After purification, polypeptides may be exchanged into differentbuffers and/or concentrated by any of a variety of methods known to theart, including, but not limited to, filtration and dialysis.

The purified polypeptide is preferably at least 85% pure, or preferablyat least 95% pure, and most preferably at least 98% pure. Regardless ofthe exact numerical value of the purity, the polypeptide is sufficientlypure for use as a pharmaceutical product.

A platform manufacturing process was used to prepare anti-PCSK9Adnectin. The anti-PCSK9 Adnectin is produced in Escherichia coli (E.coli). E. coli BLR (DE3) cells were transformed with expression vector(pET9d/ATI001173) which produces the protein in a soluble formintracellularly. The recombinant strain is grown in stirred tankfermentors. At the end of fermentation the cells are collected, lysed,and clarified in preparation for purification. ATI001173 is a non-histagged version of ATI001114. The purified anti-PCSK9 Adnectin isconjugated to a 40 kDa branched methoxyPEG using a maleimide linker. Theconjugated material is subsequently repurified to remove free PEG, freeanti-PCSK9 Adnectin and product related impurities. Quality controltesting is performed on the bulk drug substance.

Therapeutic In Vivo Uses

The application describes anti-PCSK9 Adnectin useful in the treatment ofatherosclerosis, hypercholesterolemia and other cholesterol relateddiseases. The application also describes methods for administeringanti-PCSK9 Adnectin to a subject. The subject can be a human. Theanti-PCSK9 Adnectin can be pharmaceutically acceptable to a mammal, inparticular a human. A “pharmaceutically acceptable” polypeptide refersto a polypeptide that is administered to an animal without significantadverse medical consequences, such as essentially endotoxin free, orvery low endotoxin levels.

Formulation and Administration

The application further provides pharmaceutically acceptablecompositions comprising the anti-PCSK9 Adnectin or fusion proteinsthereof described herein, wherein the composition is essentiallyendotoxin free. Therapeutic formulations comprising anti-PCSK9 Adnectinor fusions thereof are prepared for storage by mixing the describedpolypeptide having the desired degree of purity with optionalphysiologically acceptable carriers, excipients or stabilizers (Osol,A., Remington's Pharmaceutical Sciences, 16th Edition (1980)), in theform of aqueous solutions, lyophilized or other dried formulations.Acceptable carriers, excipients, or stabilizers are nontoxic torecipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyidimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrans; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as Tween, PLURONIC® or polyethylene glycol (PEG).

The formulations herein may also contain more than one active compoundas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. Such molecules are suitably present in combination in amountsthat are effective for the purpose intended.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

The skilled artisan will understand that the dosage of each therapeuticagent will be dependent on the identity of the agent.

For therapeutic applications, the anti-PCSK9 Adnectin or a fusionprotein comprising an anti-PCSK9 Adnectin is administered to a subject,in a pharmaceutically acceptable dosage form. They can be administeredintravenously as a bolus or by continuous infusion over a period oftime, or by subcutaneous routes. Suitable pharmaceutically acceptablecarriers, diluents, and excipients are well known and can be determinedby those of skill in the art as the clinical situation warrants.Examples of suitable carriers, diluents and/or excipients include: (1)Dulbecco's phosphate buffered saline, (2) 0.9% saline (0.9% w/v NaCl),and (3) 5% (w/v) dextrose.

The method described herein can be practiced in vitro, in vivo, or exvivo.

Administration of anti-PCSK9 Adnectin or a fusion thereof, and one ormore additional therapeutic agents, whether co-administered oradministered sequentially, may occur as described above for therapeuticapplications. Suitable pharmaceutically acceptable carriers, diluents,and excipients for co-administration will be understood by the skilledartisan to depend on the identity of the particular therapeutic agentbeing administered.

When present in an aqueous dosage form, rather than being lyophilized,the protein typically will be formulated at a concentration of about 0.1mg/ml to 100 mg/ml, although wide variation outside of these ranges ispermitted. For the treatment of disease, the appropriate dosage ofanti-PCSK9 Adnectin or a fusion thereof will depend on the type ofdisease to be treated, the severity and course of the disease, whetherthe protein is administered for preventive or therapeutic purposes, thecourse of previous therapy, the patient's clinical history and responseto the protein, and the discretion of the attending physician. Theprotein is suitably administered to the patient at one time or over aseries of treatments.

Fusions of Serum Albumin Binding Adnectin (SABA)

In certain aspects, the application provides fusion proteins comprisinganti-PCSK9 Adnectin fused to a ¹⁰Fn3 domain that binds to human serumalbumin (a Serum Albumin Binding Adnectin (¹⁰Fn3 domain) or SABA). Suchfusion proteins have extended serum half lives in the presence ofalbumin relative to the anti-PCSK9 Adnectin alone (e.g., not conjugatedto a PK moiety).

¹⁰Fn3 domains are cleared rapidly from circulation via renal filtrationand degradation due to their small size of ˜10 kDa (t_(1/2=15)-45minutes in mice; 1-3 hours in monkeys). Fusion of a ¹⁰Fn3 domain, suchas an anti-PCSK9 Adnectin, to a second polypeptide comprising a ¹⁰Fn3domain that binds specifically to serum albumin, e.g., human serumalbumin (HSA), may be used to prolong the t_(1/2) of the anti-PCSK9Adnectin.

In certain embodiments, the serum half-life of the anti-PCSK9 Adnectinfused to the SABA is increased relative to the serum half-life of theanti-PCSK9 Adnectin when not conjugated to the SABA. In certainembodiments, the serum half-life of the SABA fusion is at least 20, 40,60, 80, 100, 120, 150, 180, 200, 400, 600, 800, 1000, 1200, 1500, 1800,1900, 2000, 2500, or 3000% longer relative to the serum half-life of theanti-PCSK9 Adnectin when not fused to the SABA. In other embodiments,the serum half-life of the SABA fusion is at least 1.5-fold, 2-fold,2.5-fold, 3-fold, 3.5 fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold,8-fold, 10-fold, 12-fold, 13-fold, 15-fold, 17-fold, 20-fold, 22-fold,25-fold, 27-fold, 30-fold, 35-fold, 40-fold, or 50-fold greater than theserum half-life of the anti-PCSK9 Adnectin when not fused to the SABA.In some embodiments, the serum half-life of the SABA fusion is at least10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 50hours, 60 hours, 70 hours, 80 hours, 90 hours, 100 hours, 110 hours, 120hours, 130 hours, 135 hours, 140 hours, 150 hours, 160 hours, or 200hours.

In certain embodiments, the serum albumin binding portion of the SABAfusion protein binds to HSA with a K_(D) of less than 3 uM, 2.5 uM, 2uM, 1.5 uM, 1 uM, 500 nM, 100 nM, 50 nM, 10 nM, 1 nM, 500 pM, 100 pM, 50pM or 10 pM. In certain embodiments, the serum albumin binding portionof the SABA fusion proteins bind to HSA with a K_(D) of less than 3 uM,2.5 uM, 2 uM, 1.5 uM, 1 uM, 500 nM, 100 nM, 50 nM, 10 nM, 1 nM, 500 pM,100 pM, 50 pM or 10 pM at a pH range of 5.5 to 7.4 at 25° C. or 37° C.In some embodiments, the serum albumin binding portion of the SABAfusion proteins bind more tightly to HSA at a pH less than 7.4 ascompared to binding at pH 7.4.

Accordingly, the SABA fusion molecules described herein are useful forincreasing the half-life of anti-PCSK9 Adnectin by creating a fusionbetween anti-PCSK9 Adnectin and the SABA. Such fusion molecules may beused to treat conditions which respond to the biological activity ofPCSK9. The use of the SABA fusion molecules in diseases caused by thedysregulation of PCSK9 is contemplated.

The fusion may be formed by attaching anti-PCSK9 Adnectin to either endof the SABA molecule, i.e., SABA-anti-PCSK9 Adnectin or anti-PCSK9Adnectin-SABA arrangements.

HSA has a serum concentration of 600 μM and a t_(1/2) of 19 days inhumans. The extended t_(1/2) of HSA has been attributed, in part, to itsrecycling via the neonatal Fc receptor (FcRn). HSA binds FcRn in apH-dependent manner after endosomal uptake into endothelial cells; thisinteraction recycles HSA back into the bloodstream, thereby shunting itaway from lysosomal degradation. FcRn is widely expressed and therecycling pathway is thought to be constitutive. In the majority of celltypes, most FcRn resides in the intracellular sorting endosome. HSA isreadily internalized by a nonspecific mechanism of fluid-phasepinocytosis and rescued from degradation in the lysosome by FcRn. At theacidic pH found in the endosome, HSA's affinity for FcRn increases (5 μMat pH 6.0). Once bound to FcRn, HSA is shunted away from the lysosomaldegradation pathway, transcytosed to and released at the cell surface.

In certain embodiments, the serum albumin binding portion of the SABAfusion proteins described herein may also bind serum albumin from one ormore of monkey, rat, or mouse. In certain embodiments, the HSA bindingportion of the SABA fusion proteins described herein bind to rhesusserum albumin (RhSA) or cynomolgus monkey serum albumin (CySA) with aK_(D) of less than 3 uM, 2.5 uM, 2 uM, 1.5 uM, 1 uM, 500 nM, 100 nM, 50nM, 10 nM, 1 nM, 500 pM or 100 pM.

In certain embodiments, the serum albumin binding portion of the SABAfusion proteins described herein bind to domain I and/or domain II ofHSA. In one embodiment, the HSA binding portion of the SABA fusionproteins described herein do not bind to domain III of HSA.

In certain embodiments, the serum albumin binding portion of the SABAfusion proteins comprises a sequence having at least 40%, 50%, 60%, 70%,75%, 80% or 85% identity to the wild-type ¹⁰Fn3 domain (SEQ ID NO: 1).In one embodiment, at least one of the BC, DE, or FG loops is modifiedrelative to the wild-type ¹⁰Fn3 domain. In another embodiment, at leasttwo of the BC, DE, or FG loops are modified relative to the wild-type¹⁰Fn3 domain. In another embodiment, all three of the BC, DE, and FGloops are modified relative to the wild-type ¹⁰Fn3 domain. In otherembodiments, a SABA comprises a sequence having at least 40%, 50%, 60%,70%, 75%, 80%, 85%, 90%, or 95% identity to any one of the 26 core SABAsequences shown in Table 6 (i.e., SEQ ID NO: 334, 338, 342, 346, and348-370) or any one of the extended SABA sequences shown in Table 6(i.e., SEQ ID NO: 420-447, minus the 6×HIS tag).

In certain embodiments, the serum binding Adnectins based on the ¹⁰Fn3scaffold can be defined generally by the following sequence:

(SEQ ID NO: 328)EVVAAT(X)_(a)SLLI(X)_(x)YYRITYGE(X)_(b)QEFTV(X)_(y)ATI(X)_(c)DYTITVYAV(X)_(z)ISINYRT

In certain embodiments, the serum binding Adnectins based on the ¹⁰Fn3scaffold can be defined generally by the sequence:

(SEQ ID NO: 329)EVVAATPTSLLI(X)_(x)YYRITYGETGGNSPVQEFTV(X)_(y)ATISGLKPGVDYTITVYAV(X)_(z)ISINYRT

As described herein for anti-PCSK9 Adnectins, SEQ ID NOs: 328 and 329can be defined and applied to SABA molecules in the same way. Inexemplary embodiments, the BC, DE, and FG loops as represented by(X)_(x), (X)_(y), and (X)_(z), respectively, are replaced withpolypeptides comprising the BC, DE and FG loop sequences from any of theHSA binders shown in Table 6 below (i.e., SEQ ID NOs: 330, 334, 338,342, 346, and 348-370 in Table 6). In certain embodiments, the BC, DE,or FG loop sequences shown in Table 6 may contain one or more additionalresidues flanking the N- and/or C-termini. In particular, the BC loopmay contain an SW at the N-terminus of the BC loop sequences shown inTable 6 when replacing (X)_(x) in SEQ ID NO: 328. Similarly, the DE loopmay contain a P preceding loop DE and the residue T following loop DEwhen replacing (X)_(y) in SEQ ID NO: 328. The FG loop may contain a Pfollowing the FG loop when replacing (X)_(z) in SEQ ID NO: 328. Forexample, SEQ ID NO: 330 indicates that the BC, DE, and FG loops compriseHSYYEQNS (SEQ ID NO: 638), YSQT (SEQ ID NO: 639), and YGSKYYY (SEQ IDNO: 640), respectively. However, when replacing (X)_(x), (X)_(y), and(X)_(z) in SEQ ID NO: 328, i.e., the BC, DE and FG loops, the (X)_(x)sequence may be SWHSYYEQNS (SEQ ID NO: 641), the (X)_(y) sequence may bePYSQTT (SEQ ID NO: 642), and the (X)_(z) sequence may be YGSKYYYP (SEQID NO: 643).

In certain embodiments, a SABA for use in a fusion as described hereinmay comprise the sequence as set forth in SEQ ID NO: 328 or 329, whereinthe BC, DE, and FG loops as represented by (X)_(x), (X)_(y), and(X)_(z), respectively, are replaced with a respective set of specifiedBC, DE, and FG loops from any of the 26 core SABA sequences (i.e., SEQID NOs: 330, 334, 338, 342, 346, and 348-370 in Table 6), or sequencesat least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the BC,DE and FG loop sequences of the 26 core SABA sequences. In exemplaryembodiments, a SABA as described herein is defined by SEQ ID NO: 329 andhas a set of BC, DE and FG loop sequences from any of the 26 core SABAsequences (i.e., SEQ ID NOs: 330, 334, 338, 342, 346, and 348-370 inTable 6), optionally with the N- and/or C-terminal additions to the loopsequences as described above. For example, SABA1 has the core sequenceset forth in SEQ ID NO: 330 and comprises BC, DE, and FG loops as setforth in SEQ ID NO: 331-333, respectively. Therefore, a SABA based onthe SABA1 core may comprise SEQ ID NO: 328 or 329, wherein (X)_(x)comprises SEQ ID NO: 331, (X)_(y) comprises SEQ ID NO: 332, and (X)_(z)comprises SEQ ID NO: 333. In some embodiments, the sequences thatreplace (X)_(x), (X)_(y), and (X)_(z) comprise additional residue(s) oneither or both ends of the loops as described above. Similar constructsare contemplated utilizing the set of BC, DE and FG loops from the otherSABA core sequences. The scaffold regions of such SABA may compriseanywhere from 0 to 20, from 0 to 15, from 0 to 10, from 0 to 8, from 0to 6, from 0 to 5, from 0 to 4, from 0 to 3, from 0 to 2, or from 0 to 1substitutions, conservative substitutions, deletions or additionsrelative to the scaffold amino acids residues of SEQ ID NO: 1. Suchscaffold modifications may be made, so long as the SABA is capable ofbinding serum albumin, e.g., HSA, with a desired K_(D).

In certain embodiments, a SABA (e.g., a SABA core sequence or a sequencebased thereon as described above) may be modified to comprise anN-terminal extension sequence and/or a C-terminal extension sequence.Exemplary extension sequences are shown in Table 6. For example, SEQ IDNO: 420 designated as SABA1.1 comprises the core SABA 1 sequence (SEQ IDNO: 330) with an N-terminal sequence MGVSDVPRDLE (SEQ ID NO: 371,designated as AdNT1), and a C-terminal sequence EIDKPSQ (SEQ ID NO: 380,designated as AdCT1). SABA1.1 further comprises a His6 tag at theC-terminus, however, it should be understood that the His6 tag iscompletely optional and may be placed anywhere within the N- orC-terminal extension sequences, or may be absent from the sequence alltogether. Further, any of the exemplary N- or C-terminal extensionsequences provided in Table 6 (SEQ ID NO: 371-395), and any variantsthereof, can be used to modify any given SABA core sequence provided inTable 6.

In certain embodiments, the C-terminal extension sequences (also called“tails”), comprise E and D residues, and may be between 8 and 50, 10 and30, 10 and 20, 5 and 10, and 2 and 4 amino acids in length. In someembodiments, tail sequences include ED-based linkers in which thesequence comprises tandem repeats of ED. In exemplary embodiments, thetail sequence comprises 2-10, 2-7, 2-5, 3-10, 3-7, 3-5, 3, 4 or 5 EDrepeats. In certain embodiments, the ED-based tail sequences may alsoinclude additional amino acid residues, such as, for example: EI, EID,ES, EC, EGS, and EGC. Such sequences are based, in part, on knownAdnectin tail sequences, such as EIDKPSQ (SEQ ID NO: 380), in whichresidues D and K have been removed. In exemplary embodiments, theED-based tail comprises an E, I or EI residues before the ED repeats.

In other embodiments, the tail sequences may be combined with otherknown linker sequences (e.g., SEQ ID NO: 396-419 in Table 6) asnecessary when designing a SABA fusion molecule.

Conjugation/Linkers

SABA fusions may be covalently or non-covalently linked. In someembodiments, a serum albumin binding ¹⁰Fn3 may be directly or indirectlylinked to an anti-PCSK9 Adnectin via a polypeptide linker. Suitablelinkers for joining Fn3 domains are those which allow the separatedomains to fold independently of each other forming a three dimensionalstructure that permits high affinity binding to a target molecule.

The disclosure provides a number of suitable linkers that meet theserequirements, including glycine-serine based linkers, glycine-prolinebased linkers, as well as the linker having the amino acid sequencePSTSTST (SEQ ID NO: 416). The Examples described herein demonstrate thatFn3 domains joined via polypeptide linkers retain their target bindingfunction. In some embodiments, the linker is a glycine-serine basedlinker. These linkers comprise glycine and serine residues and may bebetween 8 and 50, 10 and 30, and 10 and 20 amino acids in length.Examples include linkers having an amino acid sequence (GS)₇ (SEQ ID NO:403), G(GS)₆ (SEQ ID NO: 398), and G(GS)₇G (SEQ ID NO: 400). Otherlinkers contain glutamic acid, and include, for example, (GSE)₅ (SEQ IDNO: 405) and GGSE GGSE (SEQ ID NO: 409). Other exemplary glycine-serinelinkers include (GS)₄ (SEQ ID NO: 402), (GGGGS)₇ (SEQ ID NO: 411),(GGGGS)₅ (SEQ ID NO: 412), and (GGGGS)₃G (SEQ ID NO: 413). In someembodiments, the linker is a glycine-proline based linker. These linkerscomprise glycine and proline residues and may be between 3 and 30, 10and 30, and 3 and 20 amino acids in length. Examples include linkershaving an amino acid sequence (GP)₃G (SEQ ID NO: 414), (GP)₅G (SEQ IDNO: 415), and GPG. In other embodiments, the linker may be aproline-alanine based linker having between 3 and 30, 10 and 30, and 3and 20 amino acids in length. Examples of proline alanine based linkersinclude, for example, (PA)₃ (SEQ ID NO: 417), (PA)₆ (SEQ ID NO: 418) and(PA)₉ (SEQ ID NO: 419). It is contemplated, that the optimal linkerlength and amino acid composition may be determined by routineexperimentation in view of the teachings provided herein. In someembodiments, an anti-PCSK9 Adnectin is linked to a SABA via apolypeptide linker having a protease site that is cleavable by aprotease in the blood or target tissue. Such embodiments can be used torelease an anti-PCSK9 Adnectin for better delivery or therapeuticproperties or more efficient production.

Additional linkers or spacers, may be introduced at the C-terminus of aFn3 domain between the Fn3 domain and the polypeptide linker. Additionallinkers or spacers may be introduced at the N-terminus of a Fn3 domainbetween the Fn3 domain and the polypeptide linker.

In some embodiments, an anti-PCSK9 Adnectin may be directly orindirectly linked to a SABA via a polymeric linker. Polymeric linkerscan be used to optimally vary the distance between each component of thefusion to create a protein fusion with one or more of the followingcharacteristics: 1) reduced or increased steric hindrance of binding ofone or more protein domains when binding to a protein of interest, 2)increased protein stability or solubility, 3) decreased proteinaggregation, and 4) increased overall avidity or affinity of theprotein.

In some embodiments, an anti-PCSK9 Adnectin is linked to a SABA via abiocompatible polymer such as a polymeric sugar. The polymeric sugar caninclude an enzymatic cleavage site that is cleavable by an enzyme in theblood or target tissue. Such embodiments can be used to release ananti-PCSK9 Adnectin for better delivery or therapeutic properties ormore efficient production.

Summary of Sequences

Many of the sequences referenced in “Fusions of Serum Albumin BindingAdnectin (SABA)” and “Conjugation/Linkers” sections above are summarizedin Table 6 below. Unless otherwise specified, all N-terminal extensionsare indicated with a single underline, all C-terminal tails/extensionsare indicated with a double underline, and linker sequences are boxed.Loop regions BC, DE and FG are italicized for each core SABA sequence.As described further above, the modification sequences (e.g., N or Cterminal extensions and linkers) can also be used to modify anti-PCSK9Adnectin molecules.

TABLE 6 Summary of Exemplary Sequences SEQ Sequence ID Name DescriptionSequence Exemplary Serum Albumin-Binding Adnectins (SABA) 327 ¹⁰Fn3WT WTcore human ¹⁰Fn3 EVVAATPTSLLISWDAPAVTVRYYRITYGET core domainGGNSPVQEFTVPGSKSTATISGLKPGVDYTI TVYAVTGRGDSPASSKPISINYRT 328 ¹⁰Fn3v6Generic ¹⁰Fn3 having 6 EVVAAT(X)_(a)SLLI(X)_(x)YYRITYGE(X)_(b)QEvariable loops FTV(X)_(y)ATI(X)_(c)DYTITVYAV(X)_(z)ISINY RT 329 ¹⁰Fn3v3Generic ¹⁰Fn3 having 3 EVVAATPTSLLI(X)_(x)YYRITYGETGGNSPV variable loopsQEFTV(X)_(y)ATISGLKPGVDYTITVYAV(X)_(z) ISINYRT 330 SABA1 Core 1 AdnectinEVVAATPTSLLISWHSYYEQNSYYRITYGET GGNSPVQEFTVPYSQTTATISGLKPGVDYTITVYAVYGSKYYYPISINYRT 331 SABA1BC Core 1 BC Loop HSYYEQNS 332 SABA1DECore 1 DE Loop YSQT 333 SABA1FG Core 1 FG Loop YGSKYYY 334 SABA2 Core 2Adnectin EVVAATPTSLLISWPKYDKTGHYYRITYGET GGNSPVQEFTVPTRQTTATISGLKPGVDYTITVYAVSKDDYYPHEHRPISINYRT 335 SABA2BC Core 2 BC Loop PKYDKTGH 336 SABA2DECore 2 DE Loop TRQT 337 SABA2FG Core 2 FG Loop SKDDYYPHEHR 338 SABA3Core 3 Adnectin EVVAATPTSLLISWSNDGPGLSYYRITYGETGGNSPVQEFTVPSSQTTATISGLKPGVDYTI TVYAVSYYTKKAYSAGPISINYRT 339 SABA3BCCore 3 BC Loop SNDGPGLS 340 SABA3DE Core 3 DE Loop SSQT 341 SABA3FG Core3 FG Loop SYYTKKAYSAG 342 SABA4 Core 4 Adnectin;EMVAATPTSLLISWEDDSYYSRYYRITYGET contains a scaffoldGGNSPVQEFTVPSDLYTATISGLKPGVDYTI mutation (bolded);TVYAVTYDVTDLIMHEPISINYRT scaffold-perfect version is SABA5 343 SABA4BCCore 4 BC Loop EDDSYYSR 344 SABA4DE Core 4 DE Loop SDLY 345 SABA4FG Core4 FG Loop YDVTDLIMHE 346 SABA5 Core 5 Adnectin; seeEVVAATPTSLLISWEDDSYYSRYYRITYGET description for SABA4;GGNSPVQEFTVPSDLYTATISGLKPGVDYTI corrected residue isTVYAVTYDVTDLIMHEPISINYRT bolded 347 SABA5BC Core 5 BC Loop EDDSYYSR 348SABA5DE Core 5 DE Loop SDLY 349 SABA5FG Core 5 FG Loop YDVTDLIMHE 350SABA6 Core 6 Adnectin EVVAATPTSLLISWYMDEYDVRYYRITYGETGGNSPVQEFTVPNYYNTATISGLKPGVDYTI TVYAVTRIKANNYMYGPISINYRT 351 SABA7 Core7 Adnectin EVVAATPTSLLISWNHLEHVARYYRITYGETGGNSPVQEFTVPEYPTTATISGLKPGVDYTI TVYAVTITMLKYPTQSPISINYRT 352 SABA8 Core8 Adnectin EVVAATPTSLLISWGHYRRSGHYYRITYGETGGNSPVQEFTVDPSSYTATISGLKPGVDYTI TVYAVSKDDYYPHEHRPISINYRT 353 SABA9 Core9 Adnectin EVVAATPTSLLISWDASHYERRYYRITYGETGGNSPVQEFTVPRYHHTATISGLKPGVDYTI TVYAVTQAQEHYQPPISINYRT 354 SABA10 Core10 Adnectin EVVAATPTSLLISWNSYYHSADYYRITYGETGGNSPVQEFTVPYPPTTATISGLKPGVDYTI TVYAVYSAKSYYPISINYRT 355 SABA11 Core 11Adnectin EVVAATPTSLLISWSKYSKHGHYYRITYGET GGNSPVQEFTVPSGNATATISGLKPGVDYTITVYAVEDTNDYPHTHRPISINYRT 356 SABA12 Core 12 AdnectinEVVAATPTSLLISWHGEPDQTRYYRITYGET GGNSPVQEFTVPPYRRTATISGLKPGVDYTITVYAVTSGYTGHYQPISINYRT 357 SABA13 Core 13 AdnectinEVVAATPTSLLISWSKYSKHGHYYRITYGET GGNSPVQEFTVDPSSYTATISGLKPGVDYTITVYAVSKDDYYPHEHRPISINYRT 358 SABA14 Core 14 AdnectinEVVAATPTSLLISWYEPYTPIHYYRITYGET GGNSPVQEFTVPGYYGTATISGLKPGVDYTITVYAVYGYYQYTPISINYRT 359 SABA15 Core 15 AdnectinEVVAATPTSLLISWSKYSKHGHYYRITYGET GGNSPVQEFTVPSGNATATISGLKPGVDYTITVYAVSDDNKYYHQHRPISINYRT 360 SABA16 Core 16 AdnectinEVVAATPTSLLISWGHYRRSGHYYRITYGET GGNSPVQEFTVDPSSYTATISGLKPGVDYTITVYAVSKDDYYPHEHRPISINYRT 361 SABA17 Core 17 AdnectinEVVAATPTSLLISWSKYSKHGHYYRITYGET GGNSPVQEFTVPSGNATATISGLKPGVDYTITVYAVEDTNDYPHTHRPISINYRT 362 SABA18 Core 18 AdnectinEVVAATPTSLLISWYEPGASVYYYRITYGET GGNSPVQEFTVPSYYHTATISGLKPGVDYTITVYAVYGYYEYEPISINYRT 363 SABA19 Core 19 AdnectinEVVAATPTSLLISWQSYYAHSDYYRITYGET GGNSPVQEFTVPYPPQTATISGLKPGVDYTITVYAVYAGSSYYPISINYRT 364 SABA20 Core 20 AdnectinEVVAATPTSLLISWGHYRRSGHYYRITYGET GGNSPVQEFTVDPSSYTATISGLKPGVDYTITVYAVSKDDYYPHEHRPISINYRT 365 SABA21 Core 21 AdnectinEVVAATPTSLLISWPEPGTPVYYYRITYGET GGNSPVQEFTVPAYYGTATISGLKPGVDYTITVYAVYGYYDYSPISINYRT 366 SABA22 Core 22 AdnectinEVVAATPTSLLISWYRYEKTQHYYRITYGET GGNSPVQEFTVPPESGTATISGLKPGVDYTITVYAVYAGYEYPHTHRPISINYRT 367 SABA23 Core 23 AdnectinEVVAATPTSLLISWVKSEEYYRYYRITYGET GGNSPVQEFTVPYYVHTATISGLKPGVDYTITVYAVTEYYYAGAVVSVPISINYRT 368 SABA24 Core 24 AdnectinEVVAATPTSLLISWYDPYTYGSYYRITYGET GGNSPVQEFTVGPYTTTATISGLKPGVDYTITVYAVSYYYSTQPISINYRT 369 SABA25 Core 25 AdnectinEVVAATPTSLLISWSNDGPGLSYYRITYGET GGNSPVQEFTVPSSQTTATISGLKPGVDYTITVYAVSYYTKKAYSAGPISINYRT 370 SABA26 Core 26 AdnectinEVVAATPTSLLISWPDPYYKPDYYRITYGET GGNSPVQEFTVPRDYTTATISGLKPGVDYTITVYAVYSYYGYYPISINYRT Exemplary Adnectin N-Terminal Extension Sequences371 AdNT1 Exemplary leader MGVSDVPRDL 372 AdNT2 Exemplary leaderGVSDVPRDL 373 AdNT3 Exemplary leader VSDVPRDL 374 AdNT4 Exemplary leaderSDVPRDL 375 AdNT5 Exemplary leader DVPRDL 376 AdNT6 Exemplary leaderVPRDL 377 AdNT7 Exemplary leader PRDL 378 AdNT8 Exemplary leader RDL 379AdNT9 Exemplary leader DL Exemplary Adnectin C-Terminal ExtensionSequences 380 AdCT1 Exemplary tail EIDKPSQ 381 AdCT2 Exemplary tailEIDKPS 382 AdCT3 Exemplary tail EIDKPC 383 AdCT4 Exemplary tail EIDKP384 AdCT5 Exemplary tail EIDK 385 AdCT6 Exemplary tail EI 386 AdCT7Exemplary tail EIEKPSQ 387 AdCT8 Exemplary tail EIDKPSQLE 388 AdCT9Exemplary tail EIEDEDEDEDED 389 AdCT10 Exemplary tail EIEKPSQEDEDEDEDED390 AdCT11 Exemplary tail EGSGS 391 AdCT12 Exemplary tail EIDKPCQ 392AdCT13 Exemplary tail EIEKPCQ 393 AdCT14 Exemplary tail GSGC 394 AdCT15Exemplary tail EGSGC 395 AdCT16 Exemplary tail EIDKPCQLE 396 L1 G(GS)₂GGSGS 397 L2 G(GS)₄ GGSGSGSGS 398 L3 G(GS)₆ GGSGSGSGSGSGS 399 L4 G(GS)₇GGSGSGSGSGSGSGS 400 L5 G(GS)₇G GGSGSGSGSGSGSGSG 401 L6 GSGS GSGS 402 L7(GS)₄ GSGSGSGS 403 L7 (GS)₇ GSGSGSGSGSGSGS 404 L9 GS(A)9GS GSAAAAAAAAAGS405 L10 (GSE)₅ GSEGSEGSEGSEGSE 406 L11 (PAS)₅ PASPASPASPASPAS 407 L12(GSP)₅ GSPGSPGSPGSPGSP 408 L13 GS(TVAAPS)₂ GSTVAAPSTVAAPS 409 L14(GGSE)₂ GGSEGGSE 410 L15 (ST)₃G STSTSTG 411 L16 (GGGGS)₇GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSG GGGS 412 L17 (GGGGS)₅GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 413 L18 (GGGGS)₃G GGGGSGGGGSGGGGSG 414L19 (GP)₃G GPGPGPG 415 L20 (GP)₅G GPGPGPGPGPG 416 L21 P(ST)₃ PSTSTST 417L22 (PA)₃ PAPAPA 418 L23 (PA)₆ PAPAPAPAPAPA 419 L24 (PA)₉PAPAPAPAPAPAPAPAPA Exemplary Extensions to Adnectin Core Sequences 420SABA1.1 Adnectin core 1 MGVSDVPRDLEVVAATPTSLLISWHSYYEQN sequence havingAdNT1 SYYRITYGETGGNSPVQEFTVPYSQTTATIS and AdCT1 terminalGLKPGVDYTITVYAVYGSKYYYPISINYRTE sequences with His6 tag IDKPSQHHHHHH 421SABA1.2 Adnectin core 1 MGVSDVPRDLEVVAATPTSLLISWHSYYEQN sequence havingAdNT1 SYYRITYGETGGNSPVQEFTVPYSQTTATIS and AdCT8 terminalGLKPGVDYTITVYAVYGSKYYYPISINYRTE sequences IEDEDEDEDED 422 SABA1.3Adnectin core 1 MGVSDVPRDLEVVAATPTSLLISWHSYYEQN sequence having AdNT1SYYRITYGETGGNSPVQEFTVPYSQTTATIS and AdCT9 terminalGLKPGVDYTITVYAVYGSKYYYPISINYRTE sequences with His6 tagIEDEDEDEDEDHHHHHH 423 SABA2.1 Adnectin core 2MGVSDVPRDLEVVAATPTSLLISWPKYDKTG sequence having AdNT1HYYRITYGETGGNSPVQEFTVPTRQTTATIS and AdCT1 terminalGLKPGVDYTITVYAVSKDDYYPHEHRPISIN sequences with His6 tag YRTEIDKPSQHHHHHH424 SABA3.1 Adnectin core 3 MGVSDVPRDLEVVAATPTSLLISWSNDGPGL sequencehaving AdNT1 SYYRITYGETGGNSPVQEFTVPSSQTTATIS and AdCT1 terminalGLKPGVDYTITVYAVSYYTKKAYSAGPISIN sequences with His6 tag YRTEIDKPSQHHHHHH425 SABA4.1 Adnectin core 4 MGVSDVPRDLEMVAATPTSLLISWEDDSYYS sequencehaving AdNT1 RYYRITYGETGGNSPVQEFTVPSDLYTATIS and AdCT1 terminalGLKPGVDYTITVYAVTYDVTDLIMHEPISIN sequences with His6 tag YRTEIDKPSQHHHHHH426 SABA5.1 Adnectin core 5 MGVSDVPRDLEVVAATPTSLLISWEDDSYYS sequencehaving AdNT1 RYYRITYGETGGNSPVQEFTVPSDLYTATIS and AdCT1 terminalGLKPGVDYTITVYAVTYDVTDLIMHEPISIN sequences with His6 tag YRTEIDKPSQHHHHHH427 SABA6.1 Adnectin core 6 MGVSDVPRDLEVVAATPTSLLISWYMDEYDV sequencehaving AdNT1 RYYRITYGETGGNSPVQEFTVPNYYNTATIS and AdCT1 terminalGLKPGVDYTITVYAVTRIKANNYMYGPISIN sequences with His6 tag YRTEIDKPSQHHHHHH428 SABA7.1 Adnectin core 7 MGVSDVPRDLEVVAATPTSLLISWNHLEHVA sequencehaving AdNT1 RYYRITYGETGGNSPVQEFTVPEYPTTATIS and AdCT1 terminalGLKPGVDYTITVYAVTITMLKYPTQSPISIN sequences with His6 tag YRTEIDKPSQHHHHHH429 SABA8.1 Adnectin core 8 MGVSDVPRDLEVVAATPTSLLISWGHYRRSG sequencehaving AdNT1 HYYRITYGETGGNSPVQEFTVDPSSYTATIS and AdCT1 terminalGLKPGVDYTITVYAVSKDDYYPHEHRPISIN sequences with His6 tag YRTEIDKPSQHHHHHH430 SABA9.1 Adnectin core 9 MGVSDVPRDLEVVAATPTSLLISWDASHYER sequencehaving AdNT1 RYYRITYGETGGNSPVQEFTVPRYHHTATIS and AdCT1 terminalGLKPGVDYTITVYAVTQAQEHYQPPISINYR sequences with His6 tag TEIDKPSQHHHHHH431 SABA10.1 Adnectin core 10 MGVSDVPRDLEVVAATPTSLLISWNSYYHSA sequencehaving AdNT1 DYYRITYGETGGNSPVQEFTVPYPPTTATIS and AdCT1 terminalGLKPGVDYTITVYAVYSAKSYYPISINYRTE sequences with His6 tag IDKPSQHHHHHH 432SABA11.1 Adnectin core 11 MGVSDVPRDLEVVAATPTSLLISWSKYSKHG sequencehaving AdNT1 HYYRITYGETGGNSPVQEFTVPSGNATATIS and AdCT1 terminalGLKPGVDYTITVYAVEDTNDYPHTHRPISIN sequences with His6 tag YRTEIDKPSQHHHHHH433 SABA12.1 Adnectin core 12 MGVSDVPRDLEVVAATPTSLLISWHGEPDQT sequencehaving AdNT1 RYYRITYGETGGNSPVQEFTVPPYRRTATIS and AdCT1 terminalGLKPGVDYTITVYAVTSGYTGHYQPISINYR sequences with His6 tag TEIDKPSQHHHHHH434 SABA13.1 Adnectin core 13 MGVSDVPRDLEVVAATPTSLLISWSKYSKHG sequencehaving AdNT1 HYYRITYGETGGNSPVQEFTVDPSSYTATIS and AdCT1 terminalGLKPGVDYTITVYAVSKDDYYPHEHRPISIN sequences with His6 tag YRTEIDKPSQHHHHHH435 SABA14.1 Adnectin core 14 MGVSDVPRDLEVVAATPTSLLISWYEPYTPI sequencehaving AdNT1 HYYRITYGETGGNSPVQEFTVPGYYGTATIS and AdCT1 terminalGLKPGVDYTITVYAVYGYYQYTPISINYRTE sequences with His6 tag IDKPSQHHHHHH 436SABA15.1 Adnectin core 15 MGVSDVPRDLEVVAATPTSLLISWSKYSKHG sequencehaving AdNT1 HYYRITYGETGGNSPVQEFTVPSGNATATIS and AdCT1 terminalGLKPGVDYTITVYAVSDDNKYYHQHRPISIN sequences with His6 tag YRTEIDKPSQHHHHHH437 SABA16.1 Adnectin core 16 MGVSDVPRDLEVVAATPTSLLISWGHYRRSG sequencehaving AdNT1 HYYRITYGETGGNSPVQEFTVDPSSYTATIS and AdCT1 terminalGLKPGVDYTITVYAVSKDDYYPHEHRPISIN sequences with His6 tag YRTEIDKPSQHHHHHH438 SABA17.1 Adnectin core 17 MGVSDVPRDLEVVAATPTSLLISWSKYSKHG sequencehaving AdNT1 HYYRITYGETGGNSPVQEFTVPSGNATATIS and AdCT1 terminalGLKPGVDYTITVYAVEDTNDYPHTHRPISIN sequences with His6 tag YRTEIDKPSQHHHHHH439 SABA18.1 Adnectin core 18 MGVSDVPRDLEVVAATPTSLLISWYEPGASV sequencehaving AdNT1 YYYRITYGETGGNSPVQEFTVPSYYHTATIS and AdCT1 terminalGLKPGVDYTITVYAVYGYYEYEPISINYRTE sequences with His6 tag IDKPSQHHHHHH 440SABA19.1 Adnectin core 19 MGVSDVPRDLEVVAATPTSLLISWQSYYAHS sequencehaving AdNT1 DYYRITYGETGGNSPVQEFTVPYPPQTATIS and AdCT1 terminalGLKPGVDYTITVYAVYAGSSYYPISINYRTE sequences with His6 tag IDKPSQHHHHHH 441SABA20.1 Adnectin core 20 MGVSDVPRDLEVVAATPTSLLISWGHYRRSG sequencehaving AdNT1 HYYRITYGETGGNSPVQEFTVDPSSYTATIS and AdCT1 terminalGLKPGVDYTITVYAVSKDDYYPHEHRPISIN sequences with His6 tag YRTEIDKPSQHHHHHH442 SABA21.1 Adnectin core 21 MGVSDVPRDLEVVAATPTSLLISWPEPGTPV sequencehaving AdNT1 YYYRITYGETGGNSPVQEFTVPAYYGTATIS and AdCT1 terminalGLKPGVDYTITVYAVYGYYDYSPISINYRTE sequences with His6 tag IDKPSQHHHHHH 443SABA22.1 Adnectin core 22 MGVSDVPRDLEVVAATPTSLLISWYRYEKTQ sequencehaving AdNT1 HYYRITYGETGGNSPVQEFTVPPESGTATIS and AdCT1 terminalGLKPGVDYTITVYAVYAGYEYPHTHRPISIN sequences with His6 tag YRTEIDKPSQHHHHHH444 SABA23.1 Adnectin core 23 MGVSDVPRDLEVVAATPTSLLISWVKSEEYY sequencehaving AdNT1 RYYRITYGETGGNSPVQEFTVPYYVHTATIS and AdCT1 terminalGLKPGVDYTITVYAVTEYYYAGAVVSVPISI sequences with His6 tagNYRTEIDKPSQHHHHHH 445 SABA24.1 Adnectin core 24MGVSDVPRDLEVVAATPTSLLISWYDPYTYG sequence having AdNT1SYYRITYGETGGNSPVQEFTVGPYTTTATIS and AdCT1 terminalGLKPGVDYTITVYAVSYYYSTQPISINYRTE sequences with His6 tag IDKPSQHHHHHH 446SABA25.1 Adnectin core 25 MGVSDVPRDLEVVAATPTSLLISWSNDGPGL sequencehaving AdNT1 SYYRITYGETGGNSPVQEFTVPSSQTTATIS and AdCT1 terminalGLKPGVDYTITVYAVSYYTKKAYSAGPISIN sequences with His6 tag YRTEIDKPSQHHHHHH447 SABA26.1 Adnectin core 26 MGVSDVPRDLEVVAATPTSLLISWPDPYYKP sequencehaving AdNT1 DYYRITYGETGGNSPVQEFTVPRDYTTATIS and AdCT1 terminalGLKPGVDYTITVYAVYSYYGYYPISINYRTE sequences with His6 tag IDKPSQHHHHHH

EXAMPLES Example 1 Material and Methods Used Herein

High Throughput Protein Production (HTPP)

Selected binders cloned into pET9d vector and transformed into E. coliHMS174 cells were inoculated in 5 ml LB medium containing 50 μg/mLkanamycin in a 24-well format and grown at 37° C. overnight. Fresh 5 mlLB medium (50 μg/mL kanamycin) cultures were prepared for inducibleexpression by aspiration 200 μl from the overnight culture anddispensing it into the appropriate well. The cultures were grown at 37°C. until A₆₀₀ 0.6-0.9. After induction with 1 mMisopropyl-β-thiogalactoside (IPTG) the culture was expressed for 6 hoursat 30° C. and harvested by centrifugation for 10 minutes at 2750 g at 4°C.

Cell pellets (in 24-well format) were lysed by resuspension in 450 μl ofLysis buffer (50 mM NaH₂PO₄, 0.5 M NaCl, lx Complete Protease InhibitorCocktail-EDTA free (Roche), 1 mM PMSF, 10 mM CHAPS, 40 mM Imidazole, 1mg/ml lysozyme, 30 μg/ml DNAse, 2 μg/ml aprotonin, pH 8.0) and shaken atroom temperature for 1-3 hours. Lysates were clarified and re-rackedinto a 96-well format by transfer into a 96-well Whatman GF/D UNIFILTER®fitted with a 96-well, 1.2 ml catch plate and filtered by positivepressure. The clarified lysates were transferred to a 96-wellNi-Chelating Plate that had been equilibrated with equilibration buffer(50 mM NaH₂PO₄, 0.5 M NaCl, 40 mM Imidazole, pH 8.0) and was incubatedfor 5 min. Unbound material was removed by vacuum. The resin was washed2×0.3 ml/well with Wash buffer #1 (50 mM NaH₂PO₄, 0.5 M NaCl, 5 mMCHAPS, 40 mM Imidazole, pH 8.0) with each wash removed by vacuum. Priorto elution each well was washed with 50 μl Elution buffer (PBS+20 mMEDTA), incubated for 5 min and this wash was discarded by vacuum.Protein was eluted by applying an additional 100 μl of Elution buffer toeach well. After a 30 minute incubation at room temperature the plate(s)were centrifuged for 5 minutes at 200 g and eluted protein is collectedin 96-well catch plates containing 5 μl of 0.5M MgCl₂ added to thebottom of elution catch plate prior to elution. Eluted protein wasquantified using a BCA assay with SGE as the protein standard.

Midscale Expression and Purification of Insoluble Fibronectin-BasedScaffold Protein Binders

For expression of insoluble clones, the clone(s), followed by theHIS₆tag, are cloned into a pET9d (EMD Bioscience, San Diego, Calif.)vector and are expressed in E. coli HMS174 cells. Twenty ml of aninoculum culture (generated from a single plated colony) is used toinoculate 1 liter of LB medium containing 50 μg/ml carbenicillin and 34μg/ml chloramphenicol. The culture is grown at 37° C. until A₆₀₀0.6-1.0. After induction with 1 mM isopropyl-β-thiogalactoside (IPTG)the culture is grown for 4 hours at 30° C. and is harvested bycentrifugation for 30 minutes at ≥10,000 g at 4° C. Cell pellets arefrozen at −80° C. The cell pellet is resuspended in 25 ml of lysisbuffer (20 mM NaH₂PO₄, 0.5 M NaCl, 1× Complete Protease InhibitorCocktail-EDTA free (Roche), 1 mM PMSF, pH 7.4) using an ULTRA-TURRAX®homogenizer (IKA works) on ice. Cell lysis is achieved by high pressurehomogenization (≥18,000 psi) using a Model M-110S MICROFLUIDIZER®(Microfluidics). The insoluble fraction is separated by centrifugationfor 30 minutes at 23,300 g at 4° C. The insoluble pellet recovered fromcentrifugation of the lysate is washed with 20 mM sodiumphosphate/500 mMNaCl, pH7.4. The pellet is resolubilized in 6.0M guanidine hydrochloridein 20 mM sodium phosphate/500M NaCl pH 7.4 with sonication followed byincubation at 37 degrees for 1-2 hours. The resolubilized pellet isfiltered to 0.45 m and loaded onto a Histrap column equilibrated withthe 20 mM sodium phosphate/500M NaCl/6.0M guanidine pH 7.4 buffer. Afterloading, the column is washed for an additional 25 CV with the samebuffer. Bound protein is eluted with 50 mM Imidazole in 20 mM sodiumphosphate/500 mM NaCl/6.0M guan-HCl pH7.4. The purified protein isrefolded by dialysis against 50 mM sodium acetate/150 mM NaCl pH 4.5.

Midscale Expression and Purification of Soluble Fibronectin-BaseScaffold Protein Binders

For expression of soluble clones, the clone(s), followed by the HIS₆tag,were cloned into a pET9d (EMD Bioscience, San Diego, Calif.) vector andwere expressed in E. coli HMS174 cells. Twenty ml of an inoculum culture(generated from a single plated colony) was used to inoculate 1 liter ofLB medium containing 50 g/ml carbenicillin and 34 μg/ml chloramphenicol.The culture was grown at 37° C. until A₆₀₀ 0.6-1.0. After induction with1 mM isopropyl-β-thiogalactoside (IPTG), the culture was grown for 4hours at 30° C. and was harvested by centrifugation for 30 minutes at≥10,000 g at 4° C. Cell pellets were frozen at −80° C. The cell pelletwas resuspended in 25 ml of lysis buffer (20 mM NaH₂PO₄, 0.5 M NaCl, lxComplete Protease Inhibitor Cocktail-EDTA free (Roche), 1 mM PMSF, pH7.4) using an ULTRA-TURRAX® homogenizer (IKA works) on ice. Cell lysiswas achieved by high pressure homogenization (≥18,000 psi) using a ModelM-110S MICROFLUIDIZER® (Microfluidics). The soluble fraction wasseparated by centrifugation for 30 minutes at 23,300 g at 4° C. Thesupernatant was clarified via 0.45 m filter. The clarified lysate wasloaded onto a Histrap column (GE) pre-equilibrated with the 20 mM sodiumphosphate/500M NaCl pH 7.4. The column was then washed with 25 columnvolumes of the same buffer, followed by 20 column volumes of 20 mMsodium phosphate/500M NaCl/25 mM Imidazole, pH 7.4 and then 35 columnvolumes of 20 mM sodium phosphate/500M NaCl/40 mM Imidazole, pH 7.4.Protein was eluted with 15 column volumes of 20 mM sodium phosphate/500MNaCl/500 mM Imidazole, pH 7.4, fractions were pooled based on absorbanceat A₂₈₀ and were dialyzed against 1×PBS, 50 mM Tris, 150 mM NaCl, pH 8.5or 50 mM NaOAc, 150 mM NaCl, pH4.5. Any precipitate was removed byfiltering at 0.22 m.

Example 2 In Vitro Nonclinical Pharmacology

K_(D) by SPR

The binding characteristics were characterized by Surface PlasmonResonance (SPR). Human PCSK9 and Cynomolgus PCSK9 were immobilized onseparate channels on one dimension of a ProteOn XPR (Bio-Rad) chipsurfaces and exposed to 6 different concentrations of 2013E01 in theother dimension of the same SPR chip surface. This allowed kineticdetermination in the absence of regeneration. Duplicate chips were usedfor kinetic determinations of the Human and Cynomolgus PCSK9 at 25° C.Evaluation of the kinetic parameters was performed using the Langmuirinteraction model and constant parameter fitting with the ProteOnManager software.

Under these conditions, anti-PCSK9 Adnectins bound to human PCSK9 withdissociation constants (K_(D)) ranging from 80 pM to 1.6 nM and to thecyno PCSK9 with dissociation constants (K_(D)) ranging from 8 nM to 24nM (Table 7). Association rates were approximately 10⁵ M⁻¹s⁻¹, coupledwith dissociations that were typically 10⁻³-10⁻⁵ s⁻¹. For someAdnectins, the off-rates from human PCSK9 were slow (on the order of10⁻⁵ s⁻¹), which is close to the limit of detection for SPR technologiesso it is possible that these dissociation constant measurements fromhuman PCSK9 are under-estimates.

TABLE 7 SPR-Determined Kinetic Parameters for Anti-PCSK9 AdnectinsAgainst Directly Immobilized Human and Cyno PCSK9 PCSK9 k_(on) k_(off)K_(D) Clone ID Species (M⁻¹ s⁻¹) (s⁻¹) (nM) 1459D05 human 1.13E+051.80E−04  1.58 ± 0.176 2013E01 human 7.03 ± 0.1E+05 2.42 ± 0.3E−05 0.292± 0.008 2013E01 cyno 2.19E+05 1.77E−03 8.1 1922G04 human 5.41E+055.08E−05 0.094 ± 0.009 1922G04 cyno 4.65E+05 7.00E−03 15.03 2011H05human 1.18E+05 9.76E−06 0.079 ± 0.038 2011H05 cyno 1.90E+05 4.40E−0323.1  2012A04 human 2.59E+05 6.47E−05 0.251 ± 0.011 2012A04 cyno1.95E+05 4.75E−03 24.32K_(D) by BLI

The binding characteristics of Adnectins and human PCSK9 were alsodetermined by Bio-Layer Interferometry (BLI). Biotinylated human PCSK9was immobilized onto superstreptavidin sensor tips which weresubsequently immersed into wells containing diluted Adnectin for theduration of the association phase. Tips were then immersed into abuffer-only well for observation of Adnectin dissociation. Experimentswere performed in a temperature controlled environment at either 25 or37° C., and the oscillation speed was set at 1500 rpm.

Binding interaction analysis was performed using proprietary softwarefrom Fortebio (Fortebio Data Analysis Software version 6.3.0.36). Globalfits were performed for all samples, using a 1:1 binding model. Thenature of these global fits constrained all values of concentration to asingle pair of association and dissociation rates that were themselvesconstrained to each other. Affinities (K_(D)), association anddissociation rates were averaged over the various loading levels used inthe analysis. Under these conditions, Adnectins bound human PCSK9 withaffinities ranging from 200 pM to 7.5 nM, as shown in Table 8 below.Association rates ranged from 10⁴-10⁵ M⁻¹s⁻¹, and coupled withdissociations that were typically 10⁻³-10⁻⁵ s⁻¹.

TABLE 8 BLI-Determined Kinetic Parameters for PCSK9 Adnectins AgainstHuman PCSK9 Clone ID k_(on) k_(off) K_(D) or ATI# (M⁻¹ s⁻¹) (s⁻¹) (nM)2381D04 2.66E+05 ± 6.4E+04 6.90E−05 ± 6.9E−05 0.237 ± 0.08 2382D095.33E+05 1.64E−04 0.304 2382D05 5.05E+05 ± 2.0E+05 1.57E−04 ± 4.2E−050.314 ± 0.06 2381B04 3.09E+05 ± 2.1E+04 1.60E−04 ± 1.6E−04 0.527 ± 0.112382E05 4.36E+05 ± 1.6E+05 2.50E−04 ± 1.1E−05 0.604 ± 0.21 2382B094.51E+05 ± 4.7E+04 2.91E−04 ± 4.0E−05 0.656 ± 0.02 2382H03 4.71E+05 ±1.4E+05 5.57E−04 ± 2.3E−04 0.677 ± 0.02 2382C09 3.49E+05 2.33E−04 0.7572971A03 5.74E+05 4.20E−04 0.806 2382G04 4.19E+05 4.64E−04 1.11 2381G092.71E+05 2.91E−04 1.12 2451C06 3.47E+05 ± 5.4E+04 4.35E−04 ± 5.4E+04 1.27 ± 0.14 2382H10 2.88E+05 3.82E−04 1.40 2013E01 5.46E+05 8.37E−041.51 2382D03 3.54E+05 6.63E−04 1.53 2381F11 3.23E+05 5.22E−04 1.592971E02 3.55E+05 5.55E−04 1.78 2382H09 2.51E+05 4.83E−04 1.86 2451H074.48E+05 9.40E−04 2.08 2382B10 3.94E+05 9.77E−04 2.35 2382C05 3.68E+058.79E−04 2.49 2971A09 3.99E+05 1.05E−03 2.79 2382E03 2.20E+05 5.96E−042.94 2381H09 1.36E+05 4.60E−04 3.23 2381B02 2.65E+05 8.31E−04 3.292381B08 2.13E+05 9.40E−04 4.11 2382F05 2.44E+05 1.09E−03 4.54 1459D051.49E+05 ± 2.5E+04 2.02E−03 ± 2.0E−03 14.4 ± 0.2 ATI 1091 2.863E+05 8.201E−05  0.293 ATI001117 1.451E+05  5.074E−05  0.554 ATI0010575.325E+05  1.403E−04  0.255 ATI001119 8.190E+04  4.450E−04  5.296ATI001168¹ 5.829E+05  3.335E−04  0.586 ATI001175² 6.558E+05  3.522E−04 0.543 ATI 1081 8.29E+05 ± 8.1E+05 3.49E−04 ± 3E−04   0.479 ± 0.11 ATI8911.08E+05 4.03E−04 3.56 ATI1114 8.072E+04  3.952E−04  4.876 ATI11741.265E+05  9.012E−04  7.397 ¹ATI001168 is a deimmunized version of1922G04 having an R23E substitution. ²ATI001175 is a deimmunized versionof 1922G04 having an R23D substitution.

Solution Phase Affinity

KinExA

The solution affinities of ATI001081 and ATI001174 for human PCSK9 weremeasured using a Kinetic Exclusion Assay (KinExA). The relative unboundAdnectin concentrations were measured by capture on a hPCSK9 solidmatrix followed by detection with a fluorescently labeled antibody thatrecognizes the Adnectin scaffold. Due to technical limitations, thelowest concentration of Adnectins that could be tested was 1 nM. Theglobal K_(D) analyses estimate a K_(D)=70 pM (28-127 pM within a 95%confidence interval) for ATI001081 and a K_(D)=223 pM for ATI001174(range of 54-585 pM within 95% confidence interval).

TABLE 9 Solution Phase Affinity Measurements for PCSK9 AdnectinsATI001081 ATI001174 K_(D) 69.5 pM 223 pM 95% confidence interval: Kdhigh  127 pM 585 pM Kd low   28 pM  54 pM

The thermodynamics and stoichiometry of binding of Adnectins ATI001174and ATI001081 to human PCSK9 were characterized by isothermal titrationcalorimetry (ITC). Solution phase binding was measured in 25 mM HEPES,pH 7.41, 150 mM NaCl at 37° C. An average unimolecular binding constantof 1.3±0.2 nM was observed for ATI001174 and 1.4±0.4 nM for ATI001081.Detailed thermodynamic analyses are shown in Table 10 and FIG. 13. Thedifference in observed enthalpies (−3.3 kcal/mol) for the two Adnectinssuggests that ATI001174 incurs an order of magnitude reduction in itsaffinity for PCSK9 due to PEGylation that is at least partially offsetby the corresponding difference in entropy (−10.4 cal/mol·K).

TABLE 10 Stoichiometry K_(A) K_(D) ΔH ΔS Adnectin (N) (M⁻¹) (nM)(kcal/mol) (cal/mol ° C.) ATI001081 0.926 7.1E8 ± 2.1E8 1.4 ± 0.4 −26.6± 0.2 −45.4 (±30%) ATI001174* 0.857 7.5E8 ± 1.5E8 1.3 ± 0.2 −29.9 ± 0.1−55.8 *Average of 3 experimentsFluorescence Resonance Energy Transfer (FRET) Assay

Three FRET based assays were developed to determine the binding affinityand potency of PCSK9-binding Adnectins, adapted from the general methoddescribed previously by Maio et al. (See, Miao, B. et al., Meth.Enzymol., 357:180-188 (2002)). The PCSK9:EGFA FRET assay (FIGS. 2 and 3)measured the inhibition of PCSK9 binding to the low density lipoproteinreceptor (LDLR) epidermal growth factor precursor homology domain (EGFAdomain), using recombinant human PCSK9 expressed in baculovirus and asynthetic 40-mer EGFA peptide (biotinylated). EGFA has been shown torepresent the key interacting domain of LDLR with PCSK9 (Kwon, H. J. etal., Proc. Natl. Acad. Sci. USA, 105(6):1820-1825 (2008)). This assayused a PCSK9 C-terminal domain binding mAb (mAb 4H5) labeled withEu-chelate to provide FRET interaction with biotinylated EGFA throughthe streptavidin/allophycocyanin fluorophore complex.

Two other related, PCSK9-dependent FRET assays were also constructed. Inone of these assays, competitive displacement by Adnectins ofbiotinylated Adnectins—ATI000972 or ATI001125 is quantified (ATI000972results shown in FIG. 4). ATI000972 is a biotinylated version of 1459D05and ATI001125 is a biotinylated version of ATI001081. In another assay,direct binding of an Adnectin (his-tagged) to PCSK9 is assayed usinganti-his6 antibody (FIG. 5). In each of the FRET assays, human PCSK9concentration was either 1 or 5 nM. In some cases cynomolgus monkeyPCSK9 replaced hPCSK9. Table 11 summarizes the overall data from thesethree FRET assays.

TABLE 11 Summary of Adnectin Testing Data for 3 FRET Assays Using HumanPCSK9 and 1 Assay for Cyno PCSK9 Direct binding hPCSK9:EGFAhPCSK9:ATI000972 hPCSK9:ATI001125 hPCSK9 cPCSK9:EGFA IC50 IC50 IC50 EC50IC50 Clone ID (nM) (nM) (nM) (nM) (nM) 1459D05 4.0 14 nd 3.9 23001784F03 2 1.9 nd 1.6 106 1813E02 1.3 2.4 nd 2.3 118 1923B02 2.3 3.2 nd3.2 53.5 1922G04 1.2 2.0 nd 2.5 26.6 2012A04 2.1 1.2 nd 1.4 70.3 2011H052.7 1.4 nd 2.0 17.2 2013E01 1.6 1.8 nd 0.90 12.5 1922G04 (R25D) 2.5 ndnd nd 48.1 1922G04 (R25E) 3.5 nd nd nd 58.5 2382E03 2.4 nd 0.5 3.7 10.82382E05 2.8 nd 0.4 3.8 12.6 2381B08 2.6 nd 1 4.1 27.1 2381B02 2.5 nd 0.58.4 9.8 2381B04 2.4 nd 0.5 4.2 17.3 2451H07 0.2 nd 0.2 13.2 27.1 2381D042.9 nd 0.5 3.7 12 2381F11 4 nd 0.8 <1 26.5 2381G09 3.1 nd 1.1 4.5 25.22381H09 3.4 nd 0.5 4.1 20.2 2382B09 3.8 nd 0.6 4.0 37.2 2382B10 3 nd 0.53.1 18.1 2382C05 4 nd 0.9 3.4 27.2 2382C09 3.5 nd 0.6 2.9 23.9 2382D033.3 nd 0.6 3.3 11.8 2382D09 2.6 nd 0.4 3.7 13.9 2382F05 2.4 nd 0.9 3.814.6 2382G04 2.9 nd 0.5 3.6 20.1 2382H03 3.4 nd 0.3 3.8 19.8 2382H09 0.9nd 0.3 3.6 17.9 2382H10 2.6 nd 0.4 4.6 18.2 2382D02 4.5 nd 0.5 5.3 57.82451C06 1.2 nd 0.4 5.5 24.7

Cell-Based Inhibition of PCSK9 Activity by PCSK9 Adnectins

DiI-LDL Uptake Assay

Cell culture methods were developed to assay the ability of Adnectins toinhibit PCSK9 activity on the LDLR. An effective means of measuringcellular LDLR activity is through an assay for uptake of labeled LDL, asshown by Lagace, T. A. et al. (J. Clin. Invest., 116(11):2995-3005(2006)). The work further adapted a method for LDLR functional activityusing fluorescent-labeled LDL (DiI-LDL) uptake adapted from a methodoriginally shown by Teupser et al. (Biochim. Biophys. Acta,1303(3):193-198 (1996)). Cells were first preincubated with recombinanthuman PCSK9 protein (10 ug/mL, 135 nM) in the presence and absence ofAdnectins as shown. After 2 hours, the remaining LDLR activity wasassayed by incubation with DiI-LDL (5 ug) for 2 hours followed by anassessment of accumulated DiI-LDL inside the cells using high contentfluorescent microscopy and image analysis (Cellomics). FIG. 6 shows theeffect of several Adnectins to inhibit PCSK9 activity and restore LDLRfunctional activity in HepG2 cells. In this assay, the Adnectinsinhibited PCSK9 and restored DiI-LDL uptake with the following EC₅₀values: 1459D05, EC₅₀=190 nM; 1784F03, 210 nM; 2012A04, 130 nM; 2013E01,160 nM.

LDLR Depletion Assay

HepG2 cells were grown in complete media, Eagle's Minimum EssentialMedium (EMEM, ATCC®) with 10% FBS (Hyclone), and split twice a week withTrypsin 0.25% (Invitrogen). To induce upregulation of the LDL receptor,cells were incubated overnight in LPDS media [RPMI (ATCC) with 5%lipoprotein deficient serum (Intracel), 100 nM superstatin (BMS) and 50uM Sodium Mevalonate (Sigma)]. The following day, cells were trypsinizedbriefly with Trypsin 0.05% (Invitrogen) and resuspended at2×10{circumflex over ( )}6 cells per ml then aliquoted at 100 ul perwell in a V-bottom 96 well plate. In the meantime, Adnectins werepre-incubated with PCSK9 in LPDS media for an hour at 37° C. After anhour, cells were centrifuged and resuspended in 100 ul of Adnectin/PCSK9mix and incubated overnight at 37° C. The following day cells werelabeled with an antibody for LDL receptor (BAF 2148 from R&D), followedby a phycoerythrin (PE)-streptavidin conjugated secondary antibody(BD554061 from BD Pharmingen) and analyzed by FACS on the FACS Cantoll(BD). 10 nM of PCSK9 was pre-incubated for an hour with increasingconcentration of Adnectin candidates before being added to HepG2 cells.After overnight incubation LDLR level were measured by FACS and thepercentage of inhibition of PCSK9-induced LDLR depletion was graphed andEC50 determined using PRISM. 1459D05 appears to be the least potentcandidate among those tested and did not reach the maximum inhibitionwhereas the other clones reach 150-200% maximum inhibition of PCSK9(FIG. 7). A summary of the PCSK9 Adnectin in vitro pharmacology data isshown below in Table 12.

TABLE 12 Cyno cross-reactivity KD hPCSK9 K_(D) hPCSK9 K_(D) cPCSK9 CynoTm (37° C., (25° C., (25° C., PCSK9:EGFA PCSK9:GFA LDLR Depletion Clone% monomer (° C., Octet Red) ProteOn) ProteOn) FRET FRET % inhibitionEC₅₀ ID (SEC-HPLC) DSC) (nM) (nM) (nM) (EC₅₀, nM) (EC₅₀, nM) at 75 nM(nM) 1459D05 ≥95 63 14.4 1.58 >1000 >1000 5.8 66.8 >200 1813E02 ≥95 70nd nd nd 118.5 2.7 nd    16 1784F03 ≥95 65 nd nd nd 106.5 2.01 150.2  26± 13 1923B02 ≥95 73 nd 0.17 nd 53.5 2.3 178 23 ± 7 1922G04 100 83 nd0.09 15.0 26.6 1.2 105.1 10 ± 2 2013E01 100 81 nd 0.29 8.1 12.5 1.6165.5 10 ± 4 2012A04 100 84 nd 0.25 24.3 70.3 2.1 144.5 12 ± 6 2011H05100 76 nd 0.08 23.1 17.2 2.7 197.6 12 ± 5 2382D05 97 86 0.314 nd nd 57.82.6 140.5 nd 2382E03 96 83 2.94 nd nd 10.8 2.4 89.5 nd 2382E05 95 840.604 nd nd 12.6 2.8 103.5 nd 2381B02 96 68 3.29 nd nd 9.8 2.5 125.4 nd2381B04 98 77 0.527 nd nd 17.3 2.4 121.6 nd 2381B08 97 78 4.11 nd nd27.1 2.6 124.8 nd 2381D04 98 70 0.237 nd nd 12 2.9 119.2 nd 2381F11 9579 1.59 nd nd 26.5 4 110.2 nd 2381G09 96 78 1.12 nd nd 25.2 3.1 133.0 nd2381H09 77 63 3.23 nd nd 20.2 3.4 94.4 nd 2382B09 99 88 0.656 nd nd 37.23.8 105.0 nd 2382B10 99 82 2.35 nd nd 18.1 3 100.2 nd 2382C05 97 85 2.49nd nd 27.2 4 105.3 nd 2382C09 96 85 0.757 nd nd 23.9 3.5 121.7 nd2382D03 97 84 1.53 nd nd 11.8 3.3 80.4 nd 2382D09 93 84 0.304 nd nd 13.92.6 109.1 nd 2382F05 99 83 4.54 nd nd 14.6 2.4 72.2 nd 2382G04 95 811.11 nd nd 20.1 2.9 146.1 nd 2382H03 96 85 0.677 nd nd 19.8 3.4 102.1 nd2382H09 95 81 1.86 nd nd 17.9 0.9 101.2 nd 2382H10 97 87 1.40 nd nd 18.22.6 118.6 nd 2451B06 nd nd nd nd nd 57.8 4.5 92.2 nd 2451C06 96 87 1.27nd nd 24.7 1.2 89.4 nd 2451H07 97 87 2.08 nd nd 27.1 0.2 88.8 nd

Example 3 In Vivo Pharmacodynamic Effects of PCSK9 Adnectins

Human PCSK9 Transgenic Mouse Models

PCSK9 Adnectins exhibited pharmacodynamic effects in vivo in twodifferent human transgenic mouse models. One mouse model overexpresseshuman PCSK9 levels markedly and exhibits hypercholesterolemia as aresult (Lagace, T. A. et al., J. Clin. Invest., 116(11):2995-3005(2006)). The other mouse model is a genomic hPCSK9 transgenic(BAC-transgenic) which is regulated in liver similarly to mouse PCSK9and which expresses near human-normal levels of hPCSK9 in plasma. Forthese studies, ELISA assays using species-specific, site-specificlabeled antibodies and Adnectins were developed to measure plasmaunbound human PCSK9 levels (i.e., hPCSK9 not complexed with theadministered Adnectin) as an index of target engagement.

Single doses of PCSK9 Adnectins in PEGylated form were injectedintraperitoneally into the overexpresser hPCSK9 transgenic mouse modelat the doses shown in FIGS. 8-9. PBS or dialysate samples were alsoinjected as controls. Adnectin 1459D05-PEG (100 mg/kg intraperitoneal)treatment rapidly decreased plasma total cholesterol (FIG. 8A) and LDL-C(not shown) to >35% below baseline in 4 hr. Cholesterol levels inAdnectin treated mice remained below control levels throughout the 48 hrtest period. This was accompanied by a sharp decrease in circulatinglevels of unbound hPCSK9 in the Adnectin treated transgenic mice (FIG.8B). Western blots of liver taken at 6 hours in parallel studies showedthat LDLR protein levels were increased ˜2-fold in Adnectin-treated mice(not shown). In further studies in this transgenic mouse model,ATI001114 was administered at 10 or 60 mg/kg. A marked, dose-dependent,rapid lowering of plasma cholesterol was seen, concomitant with adose-related reduction in unbound hPCSK9 levels (FIG. 9). These studiesrepresent in vivo proof-of-concept for PCSK9 Adnectins as effectivecholesterol lowering agents in a hypercholesterolemic human transgenicPCSK9 mouse model.

In vivo studies were conducted in the normal expresser hPCSK9 transgenicmouse model. Injection of single doses of 1459D05-PEG or ATI001114 (5mg/kg) resulted in rapid and strong decreases in unbound hPCSK9 levelsin plasma (FIG. 10). This pharmacodynamic effect on unbound hPCSK9 wasmore pronounced following ATI001114 compared to 1459D05-PEG, withgreater magnitude and duration of effect observed for the higheraffinity/potency Adnectin. A further study of dose dependency revealedthat that the 50% inhibitory dose (ED50) was less than 0.1 mg/kg forATI001114 at time points from 3 to 48 hours post-dose, as seen in FIG.11. These findings in a normal expresser transgenic mouse model showthat PCSK9 inhibitory Adnectins exhibit marked, affinity-dependent anddose-dependent effects on pharmacodynamic endpoints which are correlatedwith LDLR regulation and LDL cholesterol lowering.

Cynomolgus Monkeys

A pharmacodynamic study was conducted in normal lean cynomolgus monkeys.Adnectin ATI001114 was administered to cynos intravenously at 5 mg/kg,and plasma samples were collected at time intervals for LDL-C assay andpharmacokinetic assessment. A single dose of ATI001114 rapidly loweredplasma LDL-C levels to >50% vs. baseline (or vs. PBS control group)within 48 hours (FIG. 12). The duration of effect on LDL-C continued formore than a week with eventual return to baseline by 3 wk. This effectwas observed with both two-branched and four-branched 40 kDa PEGylatedforms of the anti-PCSK9 Adnectin (ATI001114 and ATI001211,respectively). ATI001211 is ATI001081 with a 40 kDa 4-branched NOF PEGmoiety. Total cholesterol showed a similar pattern but no effect on HDLor other metabolic parameter was observed (not shown). Pharmacokineticanalysis revealed that the plasma half-life was approximately 80-120hrs, consistent with the pharmacodynamics of LDL lowering in the cynos.These findings indicate that a PCSK9 Adnectin is efficacious andfast-acting with rapid, robust, specific effects on LDL-C lowering incynomolgus monkey model.

Example 4 In Vitro and In Vivo Pharmacological Evaluation of the PCSK9Adnectin-Fc Fusion Protein, PRD460

Production of PRD460

A vector encoding PRD460 was transfected into HEK-293 6E cells usingpolyethylenimine (PEI). The cells were grown at 37° C. for 5 days with80% humidification and 5% CO₂. The cells were then pelleted, thesupernatant was passed through a 0.22 um filter and then loaded onto aProteinA column. The column was washed with PBS and the protein waseluted with 20 mM Glycine, 150 mM NaCl pH 2.8. The eluted protein wasconcentrated and passed over a superdex200 column in 50 mM MES, 100 mMNaCl pH 5.8.

PRD460 K_(D) by SPR

The binding characteristics were characterized by Surface PlasmonResonance (SPR). Anti-human antibody was immobilized on a BIACORE® chip,and PRD460 (sequence as set forth in SEQ ID NO: 322) was captured on thechip surface. Varying concentrations of hPCSK9 were placed into the flowsolution using MgCl₂ (3 M) for chip regeneration between cycles. Forcomparison, ATI-1081 was captured on an anti-His antibody immobilized ona BIACORE® chip. Duplicate experiments for PRD460 were performed ondifferent days. Kinetic determinations were performed at 25° C.Evaluation of the kinetic parameters was performed using the 1:1 Bindingalgorithm on the BIACORE® Evaluation software.

Under these conditions, ATI-1081 bound to human PCSK9 with adissociation constant (K_(D)) of 6.7 nM at 25° C. and PRD460 bound tohuman PCSK9 with a dissociation constant (K_(D)) of 3.29+/−0.55 nM at25° C. (Table 13). The off-rate determinations using this assay formatmay be artificially limited by the off-rate of the captured ligand fromthe immobilized capture antibody, thus the assay format using directimmobilization of PCSK9 is a more accurate reflection of dissociationconstant (K_(D)) for ATI-1081.

TABLE 13 Kinetic Parameters for PRD460 and ATI- 1081 Against CapturedHuman PCSK9 ka kd KD (1/Ms) (1/s) (nM) PRD460 3.75 +/− 0.7E+04 1.21 +/−0.05E−04 3.29 +/− 0.55 ATI-1081 3.65E+04 2.45E−04 6.7PCSK9 Binding FRET Assays

Two fluorescence resonance energy transfer (FRET) based assays were usedto determine the competitive binding potency of PRD460 and otherAdnectins to hPCSK9. The PCSK9:EGFA FRET assay measures the binding ofPCSK9 to the LDLR, using a soluble epidermal growth factor precursorhomology domain-A (EGFA) peptide and recombinant human PCSK9. ThePCSK9:ATI972 FRET assay measures competitive displacement by Adnectinsof the biotinylated Adnectin, ATI-972, from PCSK9.

In the PCSK9:EGFA FRET assay (at 5 nM PCSK9), PRD460 completely andpotently displaced EGFA from the PCSK9 binding site with EC50=0.7 nM(FIG. 1, left panel). PRD460 was more potent in this assay than eitherATI-1174 (EC50=1.9 nM) or ATI-1081 (EC50=3.7 nM) (FIG. 14). The greaterapparent potency of PRD460 in this assay may be explained by bivalent(2:1) binding of Adnectin PRD460 to PCSK9 (theoretically) compared tomonovalent (1:1) binding by ATI-1081 and ATI-1174.

Using the PCSK9:ATI-972 FRET assay (at 5 nM human PCSK9), PRD460inhibited with EC50=0.3 nM, compared to 0.8 nM for ATI-1114 and 2.8 nmfor ATI-1081 (FIG. 15). These findings indicate that PRD460 potentlydisplaced the biotinylated Adnectin ATI-972 from its binding site onPCSK9. The higher potency of PRD460 relative to ATI-1081 and ATI-1174 isconsistent with bivalent binding by PRD460.

Inhibition of PCSK9-Induced LDLR Depletion in HepG2 Cells

Human PCSK9 promotes the depletion of LDLR from the surface of HepG2cells. Pre-incubation of PCSK9 with PCSK9 Adnectins inhibits PCSK9binding to LDLR and prevents the depletion of LDLR from the cellsurface. This assay was used to measure the potency of ATI-1081,ATI-1174 and PRD460 to inhibit PCSK9 induced depletion of LDLR from thecell surface.

A dilution series of PCSK9 Adnectins were pre-incubated with 10 nM humanPCSK9 for 1 hr at 37 degrees, the pre-incubated mixture was added toHepG2 cells, and the cells were incubated for 24 hours. Following thisincubation, the level of LDLR on HepG2 cells was measured using FACSanalysis. The percentage of inhibition of PCSK9-induced LDLR depletionwas calculated and graphed (FIG. 15). In this assay ATI-1081, ATI-1174,and PRD460 inhibited PCSK9 with comparable EC50's (9 nM, 8 nM and 6 nMrespectively) although a leftward-shift of the response curve wasconsistently observed for PRD460. These EC50's represent the limit ofthe assay.

PCSK9 Cell Entry Assay in HepG2 Cells

PCSK9 binding to the LDLR on the surface of hepatocytes results inco-internalization of the LDLR-PCSK9 complex during LDLR endocytosis,leading to enhanced degradation of the LDLR. A cell-based assay wasdeveloped to measure LDLR-dependent cellular entry of fluorescent PCSK9.Human PCSK9 was covalently labeled using the fluorophore ALEXAFLUOR®-647 (AF647). PCSK9-AF647 was incubated with HepG2 cells with orwithout PCSK9-Adnectins and the intracellular fluorescence wasquantified by high content fluorescent microscopy and image analysis(Cellomics). Dependence of PCSK9-AF647 cell entry on LDLR endocytosiswas established in preliminary experiments. HepG2 cells were incubatedwith 10 nM PCSK9-AF647 and varying levels of Adnectins for 4 hrs at 37degrees. In this assay, potent inhibition of PCSK9-AF647 intracellularfluorescence was observed for PRD460 (EC50=6 nM) as well as for ATI-1174(EC50=10 nM) (FIG. 16). These findings indicate that Adnectin PRD460 andATI-1174 effectively blocked the binding of PCSK9 to cell surface LDLRin a human hepatic-derived cell line in culture, thereby reducing theinternalization of PCSK9-AF647 during LDLR endocytosis.

In Vivo Transgenic Mouse Study

In vivo studies were conducted in the normal expresser hPCSK9 transgenicmouse model. Binding of Adnectins to PCSK9 in the plasma is predicted toresult in a decrease in the measured amount of unbound (free)circulating PCSK9. The decrease in unbound PCSK9 is the initialpharmacodynamic event which results in inhibition of the PCSK9-LDLRinteraction and in LDL cholesterol lowering. Administration of singledoses of PRD460 (i.p. doses from 0.6 to 18 mg/kg) to the transgenic miceresulted in rapid, strong decreases in plasma unbound hPCSK9 levels(FIG. 17). Dose-dependent decreases in unbound PCSK9 were observed withED50 <0.6 mg/kg at the 3 hr time point. These findings in the normalexpresser human PCSK9 transgenic mouse model show that PRD460 bindsstrongly and potently to circulating hPCSK9 in vivo.

In Vivo Pharmacodynamics in Cynomolgus Monkeys

The pharmacodynamic effects of PCSK9 Adnectin PRD460 were evaluated innormal lean cynomolgus monkeys. PRD460 was administered to monkeys byi.v. dosing at 15 mg/kg, and plasma samples were collected at timeintervals over 4 wks for the assay of LDL-C and free PCSK9 levels. Asingle dose of PRD460 rapidly lowered plasma LDL-C levels in themonkeys, reaching an average maximum effect of 42% of baseline LDL-C(58% reduction; n=3 monkeys) by day 3 after dosing (FIG. 18). LDL-Clevels were reduced by 50% or more for a week at this dose, remainingsignificantly below baseline for 3 wks and returning to baseline by 4wks. Total cholesterol showed a similar pattern but no effect on HDL wasobserved (not shown). Treatment with PRD460 caused an immediate drop tonear zero (below the lower limit of quantitation) in the unbound, freeform of plasma PCSK9 (FIG. 18). The free PCSK9 levels remained near thelower limits of detection for several days then gradually returned tobaseline levels by the end of 4 wks, consistent with a cause/effectrelationship with plasma LDL-C. The data indicate that plasma LDLlowering mirrored the drop in free PCSK9 levels, consistent with PCSK9inhibition regulating LDLR function following treatment with PRD460 invivo. Pharmacokinetic analysis revealed that the plasma half-life ofAdnectin PRD460 was approximately 70 hrs in this cynomolgus monkeystudy. These findings indicate that a PCSK9 Adnectin-Fc fusion proteinis highly efficacious and fast-acting with robust, specific, andlong-lasting effects on LDL-C lowering in the cynomolgus monkey model.

In Vivo Pharmacological Evaluation of the Unmodified PCSK9 Adnectin,ATI-1081

In addition to modified Adnectins containing a PK moiety (e.g.,PEGylated and Fc-fusion Adnectins), an unmodified (“naked”) PCSK9Adnectin can also be administered. Strategies for unmodified PCSK9Adnectin treatment include more frequent dosing to accommodate theshorter PK half-life, or using an extended release subcutaneousformulation to increase the length of the absorption phase and extendthe pharmacodynamic effect. Many such formulations can be envisionedincluding, as a simple example, propylene glycol/PBS solutions to delaythe rate of absorption and increase the time of exposure to the Adnectinin the circulation.

The unmodified Adnectin ATI-1081 was administered to cynomolgus monkeysi.v. at 10 mg/kg in PBS vehicle. ATI-1114 (PEGylated version of the sameAdnectin) was also administered at 1 mg/kg in PBS as a comparator.ATI-1081 elicited a rapid, transient inhibition of unbound circulatingPCSK9 levels. Within 30 minutes the extent of initial inhibitionapproached 100% (below the limits of quantitation) before returning tobaseline several hours later (FIG. 19). Concurrently, a trend to lowerLDL-C levels was observed over the first 24 hrs in the ATI-1081 treatedmonkeys (FIG. 20).

The unmodified Adnectin ATI-1081 was also administered to the normalexpresser hPCSK9 transgenic mice in a simple extended releasesubcutaneous formulation using 50:50 propylene glycol:PBS vehicle (PGvehicle) compared to PBS vehicle. This formulation is expected tomodestly delay the rate of Adnectin absorption and increase the exposuretime to ATI-1081 in the circulation, thus improving the pharmacodynamicresponse. Administration (intraperitoneal) of ATI-1081 at 1 mg/kg in PBSvehicle resulted in ˜50% lowering of unbound plasma PCSK9 at 3 hr,compared to >85% lowering for ATI-1114 at 0.3 mg/kg (FIG. 21). In asecond experiment in the transgenic mice, ATI-1081 administered bysubcutaneous injection in PG vehicle resulted in nearly equivalentdecreases in unbound LDL compared to ATI-1174, with an improved durationof effect over the first 6 hrs of the study (FIG. 22). These findingsindicate that the unmodified PCSK9 Adnectin ATI-1081 when administeredsubcutaneously in a simple extended release vehicle bound to the targethuman PCSK9 in vivo and elicited the initial pharmacodynamic response.The time dependency of the response was consistent with extended releaseprolonging the duration of effect for the unmodified PCSK9 Adnectin.

Example 5 Serum Albumin-Binding Adnectins (SABA) Example 5A. Screeningand Selection of Candidate Serum Albumin-Binding Adnectin Overview

A selection technique known as PROfusion (see e.g., Roberts et al.,Proc. Natl. Acad. Sci. USA, 94(23): 12297-12302 (1997) and WO2008/066752) was applied to a DNA library with variable regions designedinto the BC, DE and FG loops of ¹⁰Fn3. A random library of greater than10¹³ molecules was created from this design, and selection pressure wasapplied against a biotinylated form of HSA to isolate candidate serumalbumin-binding Adnectin (SABA) with desirable binding properties.

High Throughput Protein Production (HTTP) Process

The various HSA binding Adnectins were purified using a high throughputprotein production process (HTPP). Selected binders were cloned intopET9d vector containing a HIS6 tag and transformed into E. coliBL21(DE3)pLysS cells. Transformed cells were inoculated in 5 ml LBmedium containing 50 μg/mL Kanamycin in a 24-well format and grown at37° C. overnight. Fresh 5 ml LB medium (50 μg/mL Kanamycin) cultureswere prepared for inducible expression by aspirating 200 μl from theovernight culture and dispensing it into the appropriate well. Thecultures were grown at 37° C. until A₆₀₀ 0.6-0.9. After induction with 1mM isopropyl-β-thiogalactoside (IPTG), the culture was grown for another4 hours at 30° C. and harvested by centrifugation for 10 minutes at3220×g at 4° C. Cell Pellets were frozen at −80° C.

Cell pellets (in 24-well format) were lysed by resuspension in 450 μl ofLysis buffer (50 mM NaH₂PO₄, 0.5 M NaCl, 1× Complete Protease InhibitorCocktail-EDTA free (Roche), 1 mM PMSF, 10 mM CHAPS, 40 mM Imidazole, 1mg/ml lysozyme, 30 ug/ml DNAse, 2 ug/ml aprotonin, pH 8.0) and shaken atroom temperature for 1 hour. Lysates were clarified and re-racked into a96-well format by transfer into a 96-well Whatman GF/D UNIFILTER® fittedwith a 96-well, 650 μl catch plate and centrifuged for 5 minutes at200×g. The clarified lysates were transferred to a 96-well Ni-ChelatingPlate that had been equilibrated with equilibration buffer (50 mMNaH₂PO₄, 0.5 M NaCl, 10 mM CHAPS, 40 mM Imidazole, pH 8.0) and incubatedfor 5 min. Unbound material was removed. The resin was washed 2×0.3ml/well with Wash buffer #1 (50 mM NaH₂PO₄, 0.5 M NaCl, 5 mM CHAPS, 40mM Imidazole, pH 8.0). Next the resin was washed with 3×0.3 ml/well withPBS. Prior to elution each well was washed with 50 μl Elution buffer(PBS+20 mM EDTA), incubated for 5 min and this wash discarded by vacuum.Protein was eluted by applying an additional 100 ul of Elution buffer toeach well. After 30 minute incubation at room temperature the plate(s)were centrifuged for 5 minutes at 200×g and eluted protein collected in96-well catch plates containing 5 μl of 0.5M MgCl₂ affixed to the bottomof the Ni-plates. Eluted protein was quantified using a BCA Proteinassay with SGE (control Adnectin) as the protein standard. The SGEAdnectin is a wild-type ¹⁰Fn3 domain (SEQ ID NO: 1) in which integrinbinding domain (amino acids RGD at positions 78-80) have been replacedwith SGE.

HSA, RhSA and MuSA Direct Binding ELISA

For assaying direct binders to HSA, MaxiSorp plates (Nunc International,Rochester, N.Y.) were coated with 10 ug/mL HSA (Sigma, St. Louis, Mo.)in PBS at 4° C. overnight followed by blocking in casein block buffer(Thermo Scientific, Rockford, Ill.) for 1-3 hours at room temperature.For single-point screening assays, purified HTPP Adnectin were diluted1:20 in casein block buffer and allowed to bind to HSA in each well for1 hour at room temperature. For dose response assays, concentrationsranging from 0.1 nM up to 1 M were used. After washing in PBST to removeunbound Adnectins, anti-His mAb-HRP conjugate (R&D Systems, MN) diluted1:2500 in casein block buffer was added to the bound His-tagged Adnectinfor 1 hour at room temperature. Excess conjugate was removed by washingwith PBST and bound Adnectins detected using TMB detection reagents (BDBiosciences) according to the manufacturer's instructions.

Aggregation Measurement by Analytical Size Exclusion Chromatography

Size exclusion chromatography (SEC) was performed on the SABAs resultingfrom the HTPP. SEC of HTPP derived material was performed using aSUPERDEX® 200 5/150 or SUPERDEX® 75 5/150 column (GE Healthcare) on anAgilent 1100 or 1200 HPLC system with UV detection at A₂₁₄ nm and A₂₈₀nm and with fluorescence detection (excitation=280 nm, emission=350 nm).A buffer of 100 mM sodium sulfate, 100 mM sodium phosphate, 150 mMsodium chloride, pH 6.8 at appropriate flow rate of the SEC columnemployed. Gel filtration standards (Bio-Rad Laboratories, Hercules,Calif.) were used for molecular weight calibration.

The results of the SEC on the HTPP purified SABAs were shown to bepredominantly monomeric and eluted in the approximate range of 10 kDavs. globular Gel Filtration standards (BioRad).

Identification of Candidate Serum Albumin-Binding Adnectin (SABA)

As a result of the screening for HSA/RhSA/MuSA binding and biophysicalcriteria, four unique serum albumin-binding Adnectins (SABA) wereidentified and chosen to have their half-lives evaluated in mice. Inorder to carry out in vitro and in vivo characterization, midscales wereundertaken for the four SABAs. Table 6 provides the sequences oftwenty-six unique SABA core sequences identified from PROfusion,designated as SABA 1-26. SABA4 had a scaffold mutation that was fixedprior to midscaling. The scaffold-perfect version of SABA4 is SABA5.SABA4 and SABA5 have identical sequences in the BC, DE, and FG loops.

Example 5B. Production and Formulation of Candidate SABAs

Midscale Protein Production of SABAs

The selected SABAs described in Example 5A, followed by the His6 tag,were cloned into a pET 9d vector and expressed in E. coli BL21(DE3)pLysScells (see Table 6 for each His-tagged SABA sequence designated SABA1.1,SABA2.1, SABA3.1, and SABA5.1). 20 ml of an inoculum culture (generatedfrom a single plated colony) was used to inoculate 1 liter of LB mediumcontaining 50 μg/mL Kanamycin. The culture was grown at 37° C. untilA₆₀₀ 0.6-1.0. After induction with 1 mM isopropyl-β-thiogalactoside(IPTG) the culture was grown for another 4 hours at 30° C. and harvestedby centrifugation for 30 minutes at ≥10,000×g at 4° C. Cell Pellets werefrozen at −80° C. The cell pellet was resuspended in 25 mL of lysisbuffer (20 mM NaH₂PO₄, 0.5 M NaCl, 1× Complete Protease InhibitorCocktail-EDTA free (Roche), pH 7.4) using an ULTRA-TURRAX® homogenizer(IKA works) on ice. Cell lysis was achieved by high pressurehomogenization (≥18,000 psi) using a Model M-110S MICROFLUIDIZER®(Microfluidics). The soluble fraction was separated by centrifugationfor 30 minutes at 23,300×g at 4° C. The supernatant was clarified via0.45 μm filter. The clarified lysate was loaded onto a HISTRAP® column(GE) pre-equilibrated with 20 mM NaH₂PO₄, 0.5 M NaCl, pH 7.4. The columnwas then washed with 25 column volumes of 20 mM NaH₂PO₄, 0.5 M NaCl, pH7.4, followed by 20 column volumes of 20 mM NaH₂PO₄, 0.5 M NaCl, 25 mMimidazole pH 7.4, and then 35 column volumes of 20 mM NaH₂PO₄, 0.5 MNaCl, 40 mM imidazole pH 7.4. Protein was eluted with 15 column volumesof 20 mM NaH₂PO₄, 0.5 M NaCl, 500 mM imidazole pH 7.4, fractions pooledbased on absorbance at A₂₈₀ and dialyzed against 1×PBS, 50 mM Tris, 150mM NaCl pH 8.5 or 50 mM NaOAc; 150 mM NaCl; pH 4.5. Any precipitate wasremoved by filtering at 0.22 μm.

Midscale expression and purification yielded highly pure and activeAdnectins that were expressed in a soluble form and purified from thesoluble fraction of the bacterial cytosol. SEC analysis on a SUPERDEX®200 or SUPERDEX® 75 10/30GL in a mobile phase of 100 mM NaPO₄, 100 mMNaSO₄, 150 mM NaCl, pH 6.8 (GE Healthcare) demonstrated predominantlymonomeric Adnectins.

Formulation of SABA1.2

One specific SABA, SABA1.2 (SEQ ID NO: 411), was chosen for apreliminary formulation screen. SABA1.2 comprises an (ED)₅ extension onthe “core 1” sequence of ¹⁰Fn3 (see SEQ ID NO: 421 in Table 6). ForSABA1.2, a stable formulation of 10 mM succinic acid, 8% sorbitol, 5%glycine at pH 6.0 and at a product concentration of 5 mg/mL wasidentified. In this formulation the protein melting temperature was 75°C. as determined by Differential Scanning Calorimetry (DSC) using aprotein concentration of 1.25 mg/mL. The formulation providedsatisfactory physical and chemical stability at 4° C. and 25° C., withan initial aggregate level at 1.2%. After one month of stability, thelevel of aggregation was very low (1.6% at 4° C. and 3.8% at 25° C.).The protein was also stable in this formulation after five cycles offreeze-thaw as transitioned from −80° C. and −20° C. to ambienttemperature. In addition, in this formulation SABA1.2 was soluble to atleast 20 mg/mL protein concentration at 4° C. and ambient temperaturewith no precipitation or increase in aggregation.

Example 5C. Biophysical Characterization of Candidate SABAs

Size Exclusion Chromatography

Standard size exclusion chromatography (SEC) was performed on thecandidate SABAs resulting from the midscale process. SEC of midscaledmaterial was performed using a SUPERDEX® 200 10/30 or on a SUPERDEX® 7510/30 column (GE Healthcare) on an Agilent 1100 or 1200 HPLC system withUV detection at A₂₁₄ nm and A₂₈₀ nm and with fluorescence detection(excitation=280 nm, emission=350 nm). A buffer of 100 mM sodium sulfate,100 mM sodium phosphate, 150 mM sodium chloride, pH 6.8 at appropriateflow rate of the SEC column employed. Gel filtration standards (Bio-RadLaboratories, Hercules, Calif.) were used for molecular weightcalibration.

The results of the SEC on the midscaled purified SABAs showedpredominantly monomeric Adnectin and elution in the approximate range of10 kDa vs. globular Gel Filtration standards (BioRad) as showed.

Thermostability

Differential Scanning Calorimetry (DSC) analyses of the midscaled SABAswere performed to determine their respective T_(m)'s. A 1 mg/ml solutionwas scanned in a N-DSC II calorimeter (Calorimetry Sciences Corp) byramping the temperature from 5° C. to 95° C. at a rate of 1 degree perminute under 3 atm pressure. The data was analyzed vs. a control run ofthe appropriate buffer using a best fit using Orgin Software (OrginLabCorp). The results of the SEC and DSC analyses are summarized in Table14.

TABLE 14 Summary of SEC and DSC Analyses on Candidate SABAs SEC MonomerDimer DSC Clone (%) (%) (Tm) SABA1.1 92.3 7.7 63.9° C. SABA5.1 88 1270.1° C. SABA2.1 91 9 58.5° C./78.2° C. SABA3.1 99 BLD 65.2° C.

Example 5D. Characterization of Candidate SABA1 Binding to Serum Albumin

The kinetics of selected SABA clones purified from HTPP and/or midscaledmaterial described in Examples 5A and 5B were determined by capturingthe respective serum albumin (HSA/RhSA/MuSA) on the surface of aBiasensor CM5 chip and flowing a concentration series of SABAs over boththe reference flow cell and the captured albumins. In addition, bindingto albumin was carried out under various pH conditions ranging from pH5.5 to pH 7.4. HSA-binding Adnectins SABA2.1, SABA3.1, SABA4.1, andSABA1.1 cross reacted with RhSA but did not cross react with MuSA. SABA2and SABA4 binding is pH sensitive whereas clone SABA3 demonstrated pHresistance binding to HSA down to pH 6.0. SABA1.1 fits biochemicalcriteria for pH resistance and affinity/kinetics down to pH 5.5.

Domain mapping was determined by BIACORE®. Selected SABA clones purifiedfrom HTPP and/or midscaled material were determined by capturing HSA ora construct consisting of just HSA-domain I & II or HSA-domain III onthe surface of a Biasensor CM5 chip and flowing a concentration seriesof the SABAs over both the reference flow cell and the capturedalbumins. Clones SABA2 & SABA1 bound to HSA and the HSA-domain I-IIconstruct but not the HSA-domain III construct. Clones SABA3 & SABA4bound to HSA but not to either the HSA-domain I-II or HSA-domain IIIconstructs. The results are summarized in Table 15.

TABLE 15 Binding Affinity and Kinetics of Candidate SABAs (SABA1.1, 2.1,3.1 and 4.1) K_(D) K_(off) Resistant to Adnectin Target (nM) (s⁻¹) pH7.4→5.5? Epitope on HSA SABA2 HSA 33.8 +/− 20.5 (n = 6) 1.71E−04 −−−Domain I-II RhSA 63.6 4.42E−04 SABA3 HSA 863 6.82E−02 +++ Neither domainI- RhSA 431 3.37E−02 (down to pH 6.0) II nor III (interfacial?) SABA4HSA   412 +/− 8 (n = 4) 7.82E−04 −− Neither domain I- RhSA >10003.83E−03 II nor III (interfacial?) SABA1 HSA 47.2 +/− 18.2 (n = 9)4.57E−04 +++ Domain I-II RhSA  778 +/− 313 (n = 4) 5.45E−03

Example 5E. Examination of the In Vivo t_(1/2) of Candidate SABAs

The half-life of HSA in mice was determined to allow for evaluation ofHSA-binding Adnectins in mice as the HSA-binding Adnectins do not crossreact with MuSA. HSA was injected into the tail vein of approximately 6week old Ncr nude female mice at a 20 mg/kg (FIG. 23A) and 50 mg/kg dose(FIG. 23B), and the concentration of HSA in blood samples taken atintervals post-injection was determined by ELISA. The t_(1/2) of HSAinjected into mice at 20 mg/kg and 50 mg/kg were determined to be ˜24hrs and ˜20 hrs, respectively.

Half-Life Determination of SABA1-4 in Mice

One liter E. coli growth of HSA binding clones SABA1.1, SABA2.1,SABA3.1, and SABA4.1 were prepared, purified and endotoxin removed. EachSABA variant was injected into the tail vein of mice, and theconcentration in blood samples taken at intervals post-injection wasdetermined by ELISA.

The pharmacokinetic profiles of each SABA were compared in the presenceor absence of HSA in approximately 6 week old Ncr nude female mice. Themice that were co-injected with HSA had the HSA premixed with each SABA(HSA in a 3-4 molar excess) because the binding clone was selective forHSA and RhSA and did not bind the mouse serum albumin. The half-life ofSABA1.1 (clone 1318H04) in mice plasma was 0.56 hours whereas thehalf-life of SABA1.1 co-injected with HSA was 5.6 hours, a ˜10-foldincrease in half life (FIG. 24A). The half-life of SABA2.1 in miceplasma was 0.24 hours whereas the half-life of SABA2.1 co-injected withHSA was 2.8 hours, a ˜12-fold increase in half life (FIG. 24B). Thehalf-life of SABA3.1 (clone 1245H07) in mice plasma was 0.28 hourswhereas the half-life of SABA3.1 co-injected with HSA was 0.53 hours, a˜2-fold increase in half life (FIG. 24C). The half-life of SABA4.1 inmice plasma was 0.66 hours whereas the half-life of SABA4 co-injectedwith HSA was 4.6 hours, a ˜7-fold increase in half life (FIG. 24D). Asummary of the present example is shown in FIG. 25. Table 16 summarizessimilar data for SABA1.1, SABA2.1, SABA3.1, and SABA5.1; comparison ismade to half life in cyno, where available.

TABLE 16 PK (T½) CLONE Mice Cyno Comments SABA1.1 5.6 hrs 96-137 hrs T½= 96-137 hrs SABA5.1 4.6 hrs    12 hrs Poor binding affinity for RhSA.2-fold decrease in KD observed at pH <6.0 SABA2.1 2.8 hrs NA Loss ofbinding at pH <6.5 SABA3.1   32 min NA Poor T½ observed in miceHalf-Life Determination of SABA1.1 and SABA5.1 in Cynomolgus Monkeys

A three week single dose proof of concept study of SABA1.1 (FIG. 26A)and SABA5.1 (FIG. 26B) was conducted in cynomolgus monkeys to assesspharmacokinetics at a 1 mg per kg (mpk) dose IV in 2 cynomolgus monkeys.The pharmacokinetics were evaluated using a quantitative ELISA-basedassay that was developed to detect the Adnectin in plasma samples.SABA1.1 has a half-life in the range of 96-137 hours (FIG. 26A and Table17A). SABA5.1 has a half-life of approximately 12 hours and was onlymeasureable in the ELISA up to 120 hours (FIG. 26B). Table 17Asummarizes data for SABA1.1; Table 17B summarizes data for SABA5.1.

TABLE 17A SABA1.1 t½ Cmax AUCall Cl_obs Vz_obs Monkey (hrs) (μg/mL)(hr*μg/mL) (mL/hr/kg) (mL/kg) #1 95.8 9.03 673.7 1.45 200.8 #2 136.67.24 625.1 1.60 315.2

TABLE 17B SABA5.1 HL_Lambda_z Cmax AUCall Cl_obs Vz_obs (hr) (μg/mL)(hr*μg/mL) (mL/hr/kg) (mL/kg) N 2 2 2 2 2 Mean 12.186 17.358 246.8824.089 72.507 SD 1.451 3.08 36.245 0.596 19.045 Min 11.16 15.18 221.253.67 59.04 Max 13.21 19.54 272.51 4.51 85.97 CV % 11.9 17.7 14.7 14.626.3

Example 5F. Characterization of SABA1 Binding to Serum Albumin

SABA1.1 and 1.2 Binds to HSA and RhSA

SABA1.2, a “core 1” ¹⁰Fn3 comprising an (ED)₅ extension (SEQ ID NO: 421in Table 6) bound to human serum albumin (HSA) at neutral pH and 25° C.with an average association rate constant (ka) of 8.21E+03 M⁻¹s⁻¹, andan average dissociation rate constant (kd) of 4.43E−04 s⁻¹, for acalculated average K_(d) of 55.3 nM (Table 18). For rhesus serum albumin(RhSA), the measured average association rate constant was 6.6E+03M⁻¹s⁻¹, and the dissociation rate constant was 3.78E−03 s⁻¹, giving acalculated average K_(d) of 580 nM. No measurable interaction betweenSABA1.2 and mouse or rat serum albumin could be observed up to 1 μM(Table 18 and FIG. 27). At 37° C., the ka and kd increased between 2 to5-fold, leading to a ˜2-fold increase in affinity for HSA and ½ theaffinity for RhSA (Table 18).

TABLE 18 Kinetic parameters for SABA1.2 binding to albumins, in HBS-Pbuffer. Temp ka kd KD Albumin (° C.) (1/Ms) (1/s) (nM) Human 25 8.21 ±1.19E+03 4.43 ± 0.65E−04 55.3 ± 13.7 Rhesus 6.60 ± 1.18E+03 3.78 ±0.45E−03  580 ± 62.6 Mouse no observable binding Human 37 3.38E+048.15E−04 24.1 Rhesus 1.89E+04 1.85E−02 977.4 Mouse no observable binding

Additionally, a calorimetric titration was performed to determine thestoichiometry between SABA1 and HSA. For this study, SABA1.1, a “core 1”¹⁰Fn3 comprising a His6 extension (SEQ ID NO: 420 in Table 6), was used.HSA (10 μl per injection of 115 μM protein solution) was injected intothe calorimetric cell containing SABA1.1 at a concentration of 8.1 μM.The experiment was performed at 37° C. in PBS buffer pH 7.4. FIG. 28shows that SABA1.1 binds to HSA with 1:1 stoichiometry.

SABA1.2 Binds Potently to HSA at Low pH

The long half-life of albumins (e.g., t_(1/2) of HSA is 19 days) is duein large part to the fact that they are recycled from an endocyticpathway by binding to the neonatal Fc receptor, FcRn, under the low pHconditions that exist inside the endosome. As shown in Table 19 SABA1.2potently bound HSA at the endosomal pH of 5.5, suggesting that thet_(1/2) of SABA1, once bound to HSA, would also benefit from the FcRnrecycling mechanism.

TABLE 19 Comparison of Albumin Binding Kinetics at pH 7.4 and 5.5, inMES Buffer ka kd KD albumin pH (1/Ms) (1/s) (nM) Human 7.4 9.26E+033.88E−04 41.9 5.5 9.44E+03 2.70E−04 28.6 Rhesus 7.4 6.16E+03 2.95E−03479 5.5 7.57E+03 2.72E−03 359SABA1.2 Binds to Domains I and II of HSA, but not Domain III

The binding site SABA1.2 on albumin was mapped to the N-terminal domainsI or II using recombinant HSA fragments and has no detectable binding todomain III (FIG. 29). Because domain III is the domain of HSA thatprimarily interacts with FcRn, it is less likely that SABA1.2 wouldcompete for HSA binding to FcRn, again increasing the possibility offully leveraging the recycling mechanism for enhanced half-life.

Example 5G. In Vivo Pharmacology of SABA1.2

A four week single dose pre-toxicology study of SABA1.2 was conducted incynomolgus monkeys to assess pharmacokinetics and immunogenicity at twodifferent dose levels. The pharmacokinetics and immunogenicity were alsoevaluated in a three-week, single-dose pre-toxicology study thatincluded both intravenous and subcutaneous administration arms.Additionally, the pharmacokinetics of SABA1.2 was evaluated in twoseparate, single dose pre-toxicology studies in cynomolgus monkeys usinga quantitative ELISA-based assay that was developed to detect SABA1.2 inplasma samples.

SABA1.2 was administered to monkeys at 1 mpk and 10 mpk IV.Non-compartmental analyses using WINNONLIN® software were performed toevaluate pharmacokinetic parameters. As shown in FIG. 30 and theparameters described below, SABA1.2 exhibited dose-dependentpharmacokinetics in this study as determined by area under theconcentration-time curve (AUC) evaluation. The clearance (CL) forSABA1.2 at 10 mpk was 0.15 ml/hr/kg, the beta phase half-life (t_(1/2))was 143 hours, the volume of distribution (Vz) was 30 mL/kg, and totaldrug exposure (AUCall) was 5,609,457 hr*nmol/L (Table 20). The clearance(CL) for SABA1.2 at 1 mpk was 0.4 ml/hr/kg, the half-life (t_(1/2)) was124 hours, the volume of distribution (Vz) was 72 mL/kg, and total drugexposure (AUCall) was 214,636 hr*nmol/L (Table 20).

After SC or IV administration of SABA1.2, the beta-phase pharmacokineticprofiles were similar (FIG. 31). The clearance (CL) for SABA1.2 at 1 mpkIV was 0.22 ml/hr/kg, the beta phase half-life (t_(1/2)) was 125 hours,the volume of distribution (Vz) was 40 mL/kg, and total drug exposure(AUCall) was 357,993 hr*nmol/L (Table 20). The clearance (CL) forSABA1.2 at 1 mpk SC was 0.32 ml/hr/kg, the beta phase half-life(t_(1/2)) was 134 hours, the volume of distribution (Vz) was 62 mL/kg,and total drug exposure (AUCall) was 251,339 hr*nmol/L (Table 20). TheSC relative bioavailability (F) compared to IV was 0.7.

TABLE 20 Pharmacokinetic Parameters for SABA1.2 in Monkeys Study # 1 2Dose (mg/kg) 1 10 1 1 Route of i.v. i.v. i.v. s.c. administration N 3 31 2 CL (mL/hr/kg) 0.4 0.15 0.22 0.32 Vz (mL/kg) 72 30 40 62 AUCall214,636 5,609,457 357,993 251,339 (hr*nmol/L) beta T_(1/2) (h) 124 143125 134 Bioavailability n/a n/a n/a 0.7 (F)

We claim:
 1. A polypeptide comprising a fibronectin type III tenthdomain (¹⁰Fn3) wherein the ¹⁰Fn3 has a BC, DE and FG loop, and whereinthe BC loop comprises the sequence (X₁)_(z)X₂G where X₁ is any aminoacid, Z is from 6 to 9, and X₂ is Y or H, and the BC loop is selectedfrom: BC Loop SEQ ID NO SWPPPSHGYG 2 SWRPPIHAYG 3 SWDAPIHAYG 4SWDAPAHAYG 5 SWDAPAVTYG 6 SWSPPANGYG 7 SWTPPPKGYG 8 SWRPPSHAYG 9SWDPPSHAYG 10 SWEPPSHAYG 11 SWSPPSHAYG 12 SWRPPSNGHG 13 SWVPPSDDYG 14SWVPSSHAYG 15 SWDPSSHAYG 16 SWEPSSHAYG 17 SWDAPADGGYG 107 SWDAPAGDGYG108 SWAAPAGGGYG 109 SWDAPADAYG 110 SWDAPADGAYG 111 SWDAPAEGYG 112SWDAPADEAYG 113 SWDAPADGYG 114 SWDAPAGGGYG 115 SWDAPAADAYG 116SWDAPAEAGKHYG 117 SWDAPAEAYG 118 SWDAPAAAYG 119 SWDAPAGGYG 120SWDAPAAGGYG 121 SWDAPAAGYG 122 SWDAPAGAASYG 123 SWDAPAGAYG 124SWDPPAEGYG 125 SWNPPDVNYG 126 SWDAPAEGGYG 127 SWDAPAEAEAYG 131SWDAPAEGAYG 132 SWQPPAVTYG 133 SWDPPAGAYG 134 SWAPPSDAYG 135 SWDPPSDDYG301 SWDAPADDYG 302 SWDAPSDDYG 303,

wherein the DE loop comprises the sequence X₁X₁X₁X₃ where X₁ is anyamino acid and X₃ is G or S, and the DE loop is selected from: DE LoopSEQ ID NO PPGKGT 18 PIVEGT 19 PGSEGT 20 PGSKGT 21 PGSKST 22 PVGRGT 23PVGEGT 24 PIGKGT 25 PVNEGT 26 PVGVGT 27 PSSKGT 136 PVSKST 137 PVSKGT 138PVFKGT 139 PVYKGT 140 PGYKGT 141,

wherein the FG loop comprises the sequence EX₄X₁X₅X₁X₁X₆GYX₄HR (SEQ IDNO: 451) where X₁ is any amino acid, X₄ is Y or F, X₅ is Y, F, or W, andX₆ is S or A, and the FG loop is selected from: FG loop SEQ ID NOEYPYKHSGYYHRP 28 EYTFKHSGYYHRP 29 EYTYKGSGYYHRP 30 EYTYNGAGYYHRP 31EYTYIGAGYYHRP 32 EYTYEGAGYYHRP 33 EYAYNGAGYYHRP 34 EYPWKGSGYYHRP 35EFPFKWSGYYHRP 36 EFPWPHAGYYHRP 37 EYAFEGAGYYHRP 38 EYPYDYSGYYHRP 142EYPYEHSGYYHRP 143 EYPYPHSGYYHRP 144 EYTFPGAGYYHRP 145 EYPYDHSGYYHRP 146EFPYDHSGYYHRP 147 EFPYAHSGYYHRP 148 EFTFPGAGYYHRP 149 EYPYAHSGYFHRP 150EYDFPGAGYYHRP 151 EYDFPGSGYYHRP 152 EFDYPGSGYYHRP 153 EYSFPGAGYYHRP 154EFDFPGSGYYHRP 155 EFDFPGAGYYHRP 156 EFPFPGSGYYHRP 157 EFNFPGAGYYHRP 158EFTYPGSGYYHRP 159 EFPFPGAGYYHRP 160 EFNFPGSGYYHRP 161 EYPYAHAGYYHRP 162EYPYPFSGYYHRP 163 EYNFIGAGYYHRP 164 EFPYPFAGYYHRP 165 EYTYEHSGYYHRP 166EFDFVGAGYYHRP 167 EYDFAGSGYYHRP 168 EFAFPGAGYYHRP 169 EYVFPGAGYYHRP 170EYPYPFAGYYHRP 171 EYPYSHAGYYHRP 172

wherein the polypeptide comprises an amino acid sequence that is atleast 90% identical to SEQ ID NO: 209, wherein the His6 tag of SEQ IDNO: 209 is deleted, and wherein the polypeptide binds proproteinconvertase subtilisin kexin type 9 (PCSK9).
 2. The polypeptide of claim1, wherein the polypeptide comprises the amino acid sequence of SEQ IDNO: 209; wherein the amino acid sequence is truncated at the positioncorresponding to T94 of SEQ ID NO: 1, optionally with a C-terminalextension of one or more E or D residues.
 3. The polypeptide of claim 1,wherein the polypeptide comprises an amino acid sequence truncated atthe position corresponding to T94 of SEQ ID NO: 1, with a C-terminalextension of one or more E or D residues.
 4. The polypeptide of claim 1,wherein the polypeptide binds PCSK9 with a K_(D) of less than 500 nM. 5.The polypeptide of claim 1, wherein the polypeptide binds PCSK9 with aK_(D) of less than 10 nM.
 6. The polypeptide of claim 1, wherein thepolypeptide further comprises one or more pharmacokinetic (PK) moietiesselected from polyethylene glycol, sialic acid, Fc, Fc fragment,transferrin, serum albumin, a serum albumin binding protein, and a serumimmunoglobulin binding protein.
 7. The polypeptide of claim 1, whereinthe polypeptide comprises a serum albumin PK moiety.
 8. A pharmaceuticalcomposition comprising the polypeptide of claim 1 and a pharmaceuticallyacceptable carrier.
 9. The composition of claim 8, wherein thepolypeptide comprises an amino acid sequence truncated at the positioncorresponding to T94 of SEQ ID NO: 1, with a C-terminal extension of oneor more E or D residues.
 10. The composition of claim 8, wherein thepolypeptide comprises a serum albumin PK moiety.
 11. The polypeptide ofclaim 1, wherein the polypeptide binds human proprotein convertasesubtilisin kexin type 9 (PCSK9).
 12. The polypeptide of claim 1, whereinthe polypeptide comprises an amino acid sequence that is at least 95%identical to SEQ ID NO:
 209. 13. A method of treatinghypercholesterolemia in a subject comprising administering to thesubject the composition of claim
 8. 14. The method of claim 13, whereinthe subject has atherosclerosis.
 15. The method of claim 13, wherein thecomposition is administered by subcutaneous administration.
 16. Themethod of claim 13, wherein the composition is administered byintravenous administration.
 17. The method of claim 16, wherein theintravenous administration is by a bolus.
 18. The method of claim 16,wherein the intravenous administration is by continuous infusion. 19.The method of claim 14, wherein the polypeptide comprises a serumalbumin PK moiety.